Re: [ccp4bb] Refinement of heavy atoms using SOLVE or SHARP or others

[Boaz Shaanan]

Hi,
If you're using Sharp for phasing, it'll refine the positions and occupancies 
of the heavy atoms that Shelx has picked up in the first stage. I used  Sharp a 
long time ago but I'm pretty sure it still works that way.
My 2p.
Boaz

Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben Gurion University
Beer Sheva, Israel

On Oct 11, 2023 13:02, fuxingke  wrote:
Dear Colleagues,
 Reacently, for SAD experiment, I find SOLVE and SHARP can refine the 
variables of heavy atoms, such as occupancies, coordinates and thermal 
parameters. But how to use SOLVE and SHARP to refine heavy atom in command line 
in linux? I don't find a script to do it.
 Who has the scripts template that implements the refinement of heavy atoms 
using SOLVE or SHARP or others? Could you please share them with me?

Regards



Best wishes,
Fu Xingke
Institute of Physics CAS



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Re: [ccp4bb] Refinement of heavy atoms using SOLVE or SHARP or others

[Aaron Finke]

Hi,

SHARP and autoSHARP are manually run using the Sushi interface, which is not 
the easiest thing to set up. For straightforward heavy atom refinement, you can 
run both SHARP and autoSHARP on the command line. If you are familiar with 
SHARP’s input files and parameters, instructions can be found here: 
https://www.globalphasing.com/sharp/manual/appendix2.html However, if you need 
to do more difficult refinement and require more manual control over the SHARP 
parameters, the Sushi interface is vastly preferred.

If you are new to SHARP and just want to try it out on a dataset without manual 
intervention, the easiest way to do this is to use run_autoSHARP, a command 
line script that automates nearly all the setup. You supply a dataset, 
sequence, and a heavy atom list (optional, if you don’t have one, it will run 
SHELXD for you). Instructions here: 
https://www.globalphasing.com/sharp/wiki/index.cgi?RunAutoSharp

You can also run autoSHARP via CCP4, if you link your installation to it.

Best,
Aaron

From: CCP4 bulletin board  on behalf of fuxingke 

Reply-To: fuxingke 
Date: Wednesday, October 11, 2023 at 12:02
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: [ccp4bb] Refinement of heavy atoms using SOLVE or SHARP or others

Dear Colleagues,
 Reacently, for SAD experiment, I find SOLVE and SHARP can refine the 
variables of heavy atoms, such as occupancies, coordinates and thermal 
parameters. But how to use SOLVE and SHARP to refine heavy atom in command line 
in linux? I don't find a script to do it.
 Who has the scripts template that implements the refinement of heavy atoms 
using SOLVE or SHARP or others? Could you please share them with me?

Regards



Best wishes,
Fu Xingke
Institute of Physics CAS



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



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Re: [ccp4bb] Refinement of heavy atoms using SOLVE or SHARP or others

[Eleanor Dodson]

Hmm - I can give you scripts to use SHELX?
Eleanor

On Wed, 11 Oct 2023 at 11:02, fuxingke  wrote:

> Dear Colleagues,
>  Reacently, for SAD experiment, I find SOLVE and SHARP can refine the 
> variables
> of heavy atoms, such as occupancies, coordinates and thermal parameters.
> But how to use SOLVE and SHARP to refine heavy atom in command line in
> linux? I don't find a script to do it.
>  Who has the scripts template that implements the refinement of heavy
> atoms using SOLVE or SHARP or others? Could you please share them with me?
>
> Regards
>
>
>
> Best wishes,
>
> Fu Xingke
> Institute of Physics CAS
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] Refinement of ligand with alternate chemical structure

[Stuart McQuarrie]

Dear Elke,

That seems to have done the trick. Can't thank you enough!!! I'm so happy.

For future readers:
ca6 alt loc A and residue 401 70% occ
pa3 #1 alt loc B and residue 402 30% occ
pa3 #2 alt loc C and residue 403 30% occ

I could have probably done pa3 #1 and #2 as the same residue 402 as I was able 
to manipulate both in COOT but I was getting a warning with phenix.refine that 
it couldn't generate restraints for one of them so put C as 403 and that's 
worked :)

Thanks to everyone else that chipped in. It's odd that this is different from 
the way alternate protein residues are handled but there you go. Great way to 
end the week!

Kind regards,
Stuart



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Re: [ccp4bb] Refinement of ligand with alternate chemical structure

[Elke De Zitter]

Dear Stuart, 

I had some issues with different entities with the same residue number. To 
solve this, I used different numbers for the two entries and different altlocs 
(e.g. LIG-A gets number 401 and altloc A, LIG-B gets number 402 and altloc B). 
In your case you might want to call LIG-A and altloc A, LIG-A and altloc B, 
LIG-B and altloc C. Then you can restrain the occupancies to a sum of 1.0 in 
phenix.refine as described in the article shared by Pavel. This works for me 
with Phenix 1.19 and Coot 0.8.9 on Mac. 

Elke 


De: "Eleanor Dodson" <176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk> 
À: "CCP4BB"  
Envoyé: Jeudi 16 Février 2023 18:40:31 
Objet: Re: [ccp4bb] Refinement of ligand with alternate chemical structure 

Well - I made a copy of a ligand A 2001 and called it B 2001; gave each of the 
"Ligands" occupancy 0.5, mangled the B molecule a bit and ran REFMAC . 


It notes these atoms are too close but carries on refinement happily enough.. 
But when I read the structure into COOT and try to refine the A copy say it has 
a hissy fit and blows both copies out of the density... 
This is Coot version 0.9.8.7 



>From REFMAC log 
Link info given in this sectoion is for information only 
If you want to use this links then either add them into the pdb/mmcif file or 
use make link yes option 


Status Link   Mon1  At1  alt1  ch1   res1  Mon2  At2  alt2  Ch2   Res2   
distM   distI 
Unused  :.  LIG   C01   .  A 2001  LIG   C01   .  B 2001   
0.599   1.524 
Unused  :.  LIG   C01   .  A 2001  LIG   N02   .  B 2001   
1.266   1.414 
Unused  :.  LIG   C01   .  A 2001  LIG   C11   .  B 2001   
1.863   1.507 
Unused  :.  LIG   N02   .  A 2001  LIG   C01   .  B 2001   
1.799   1.414 
Unused  :.  LIG   N02   .  A 2001  LIG   N02   .  B 2001   
0.398   1.304 
Unused  :.  LIG   N02   .  A 2001  LIG   C11   .  B 2001   
0.702   1.397 
Unused  :.  LIG   C03   .  A 2001  LIG   N02   .  B 2001   
1.287   1.397 
Unused  :.  LIG   C03   .  A 2001  LIG   C03   .  B 2001   
0.699   1.490 
Unused  :.  LIG   C03   .  A 2001  LIG   C04   .  B 2001   
1.313   1.490 


On Thu, 16 Feb 2023 at 17:25, Nigel Moriarty < [ mailto:nwmoria...@lbl.gov | 
nwmoria...@lbl.gov ] > wrote: 



Stuart 
The sum of occ in these three is 1.3 and it looks like you put the occ in the 
B-factor column. 

Cheers 

Nigel 

--- 
Nigel W. Moriarty 
Building 33R0349, Molecular Biophysics and Integrated Bioimaging 
Lawrence Berkeley National Laboratory 
Berkeley, CA 94720-8235 
Email : nwmoria...@lbl.gov 
Web : [ http://cci.lbl.gov/ | CCI.LBL.gov ] 
ORCID : [ https://orcid.org/-0001-8857-9464 | orcid.org/-0001-8857-9464 
] 


On Thu, Feb 16, 2023 at 9:10 AM Stuart McQuarrie < [ 
mailto:974c6ca32bc4-dmarc-requ...@jiscmail.ac.uk | 
974c6ca32bc4-dmarc-requ...@jiscmail.ac.uk ] > wrote: 

BQ_BEGIN
Dear everyone that replied, thanks so much for the help thus far. 

Just for info I’m using Coot for Windows version 0.9.6 EL and phenix.refine 
version 1.20rc4-4416-000 

RE Eleanor: It appears my version of coot/refmac do not support separate 
ligands in the same space at <0.5 occupancy. As an experiment, I tried adding 
ATP and ADP at 0.45 occupancy to a different structure of mine. I’m able to 
real space refine in coot (so both .cifs loaded successfully) until I 
edit->merge the 2nd ligand and then the bonds look wrong and when I try RSR it 
wants to force the ligand out and explodes the molecule. 

Original message: I thought that REFMAC tolerated dual occupancies if the sum 
of the two conformers was <= 1.0? 
Eleanor 
Will test.. 

RE Pavel: I tried following that as a guide, however I’m running into an issue 
with restraints: 

I have made some progress troubleshooting the big cyclic molecule (ca6) and 
products (pA3). I re-editted the pdb; gave them the altlocs ABC; made sure they 
were same chain ID and residue number. However, when I try to real space refine 
with coot I get the error: 

“No restraints found! Non-existent or minimal description of restrained 
residues. Are you sure that you read a non-minimal mmCIF dictionary for this 
monomer? Are you sure the PDB residue name matches the dictionary residue name? 
If not, try File -> Import CIF dictionary. Alternatively, did you check that 
the atom names of the PDB file match those of the restraints? The residues in 
the chain are out of order. This can cause problems with residues selection. 
Suggest you re-order residues in increasing order. 

I definitely loaded the cifs. But it only allows me to rsr the ligand with 
altloc A. If cA6 is A then I can only rsr cA6. If pA3 is A then I can only 
refine pA3. When I delete->residue/monomer the cA6 with altloc A I can suddenly 
rsr the pA3, so both libraries must have been loaded successfully prior. I have 
included the snippet

Re: [ccp4bb] Refinement of ligand with alternate chemical structure

[Eleanor Dodson]

Well - I made a copy of a ligand A 2001 and called it B 2001; gave each of
the "Ligands" occupancy 0.5, mangled the B molecule a bit and ran REFMAC .



It notes these atoms are too close but carries on refinement happily
enough..
But when I read the structure into COOT and try to refine the A copy say it
has a hissy fit and blows both copies out of the density...
This is  Coot version 0.9.8.7


  * From REFMAC log*

*Link info given in this sectoion is for information only*

*   If you want to use this links then either add them into the
pdb/mmcif file or use make link yes option*



* Status Link   Mon1  At1  alt1  ch1   res1  Mon2  At2  alt2  Ch2
 Res2   distM   distI*

*Unused  :.  LIG   C01   .  A 2001  LIG   C01   .  B
2001   0.599   1.524*

*Unused  :.  LIG   C01   .  A 2001  LIG   N02   .  B
2001   1.266   1.414*

*Unused  :.  LIG   C01   .  A 2001  LIG   C11   .  B
2001   1.863   1.507*

*Unused  :.  LIG   N02   .  A 2001  LIG   C01   .  B
2001   1.799   1.414*

*Unused  :.  LIG   N02   .  A 2001  LIG   N02   .  B
2001   0.398   1.304*

*Unused  :.  LIG   N02   .  A 2001  LIG   C11   .  B
2001   0.702   1.397*

*Unused  :.  LIG   C03   .  A 2001  LIG   N02   .  B
2001   1.287   1.397*

*Unused  :.  LIG   C03   .  A 2001  LIG   C03   .  B
2001   0.699   1.490*

*Unused  :.  LIG   C03   .  A 2001  LIG   C04   .  B
2001   1.313   1.490*



On Thu, 16 Feb 2023 at 17:25, Nigel Moriarty  wrote:

> Stuart
>
> The sum of occ in these three is 1.3 and it looks like you put the occ in
> the B-factor column.
>
> Cheers
>
> Nigel
>
> ---
> Nigel W. Moriarty
> Building 33R0349, Molecular Biophysics and Integrated Bioimaging
> Lawrence Berkeley National Laboratory
> Berkeley, CA 94720-8235
> Email : nwmoria...@lbl.gov
> Web  : CCI.LBL.gov
> ORCID : orcid.org/-0001-8857-9464
>
>
> On Thu, Feb 16, 2023 at 9:10 AM Stuart McQuarrie <
> 974c6ca32bc4-dmarc-requ...@jiscmail.ac.uk> wrote:
>
>> Dear everyone that replied, thanks so much for the help thus far.
>>
>> Just for info I’m using Coot for Windows version 0.9.6 EL and
>> phenix.refine version 1.20rc4-4416-000
>>
>> RE Eleanor:  It appears my version of coot/refmac do not support separate
>> ligands in the same space at <0.5 occupancy. As an experiment, I tried
>> adding ATP and ADP at 0.45 occupancy to a different structure of mine. I’m
>> able to real space refine in coot (so both .cifs loaded successfully) until
>> I edit->merge the 2nd ligand and then the bonds look wrong and when I try
>> RSR it wants to force the ligand out and explodes the molecule.
>>
>> Original message: I thought that REFMAC tolerated dual occupancies if the
>> sum of the two conformers was <= 1.0?
>> Eleanor
>> Will test..
>>
>> RE Pavel: I tried following that as a guide, however I’m running into an
>> issue with restraints:
>>
>> I have made some progress troubleshooting the big cyclic molecule (ca6)
>> and products (pA3). I re-editted the pdb; gave them the altlocs ABC; made
>> sure they were same chain ID and residue number. However, when I try to
>> real space refine with coot I get the error:
>>
>> “No restraints found! Non-existent or minimal description of restrained
>> residues. Are you sure that you read a non-minimal mmCIF dictionary for
>> this monomer? Are you sure the PDB residue name matches the dictionary
>> residue name? If not, try File -> Import CIF dictionary. Alternatively, did
>> you check that the atom names of the PDB file match those of the
>> restraints? The residues in the chain are out of order. This can cause
>> problems with residues selection. Suggest you re-order residues in
>> increasing order.
>>
>> I definitely loaded the cifs. But it only allows me to rsr the ligand
>> with altloc A. If cA6 is A then I can only rsr cA6. If pA3 is A then I can
>> only refine pA3. When I delete->residue/monomer the cA6 with altloc A I can
>> suddenly rsr the pA3, so both libraries must have been loaded successfully
>> prior. I have included the snippet with the ligands just incase anyone
>> wants to see. Funnily enough phenix.refmac allowed me to refine with no
>> errors but it looks like it just ignored the pA3 as it did not properly fit
>> itself into the density the way ca6 did.
>>
>> Original Message:
>> Hi Stuart,
>> the answer, I think, is here:
>>
>> https://phenix-online.org/phenixwebsite_static/mainsite/files/newsletter/CCN_2015_07.pdf#page=12
>> Pavel
>>
>>
>> RE George: I added allow_duplicate_sequence_numbers() to my
>> coot-preferences but it didn’t seem to make a difference in my case. I
>> could already open 1EJG with no error. My issue now appears to be the
>> library file not recognising whichever ligand is not altloc A.
>>
>> Original Message:
>> Dear Stuart,
>> you have to add  allow_duplicate_sequence_numbers() to $HOME/.coot.py in
>> OSX or the appropriate place on Windows. For
>> Windows, as there is no $HOME, Coot uses .coot.py 

Re: [ccp4bb] Refinement of ligand with alternate chemical structure

[Nigel Moriarty]

Stuart

The sum of occ in these three is 1.3 and it looks like you put the occ in
the B-factor column.

Cheers

Nigel

---
Nigel W. Moriarty
Building 33R0349, Molecular Biophysics and Integrated Bioimaging
Lawrence Berkeley National Laboratory
Berkeley, CA 94720-8235
Email : nwmoria...@lbl.gov
Web  : CCI.LBL.gov
ORCID : orcid.org/-0001-8857-9464


On Thu, Feb 16, 2023 at 9:10 AM Stuart McQuarrie <
974c6ca32bc4-dmarc-requ...@jiscmail.ac.uk> wrote:

> Dear everyone that replied, thanks so much for the help thus far.
>
> Just for info I’m using Coot for Windows version 0.9.6 EL and
> phenix.refine version 1.20rc4-4416-000
>
> RE Eleanor:  It appears my version of coot/refmac do not support separate
> ligands in the same space at <0.5 occupancy. As an experiment, I tried
> adding ATP and ADP at 0.45 occupancy to a different structure of mine. I’m
> able to real space refine in coot (so both .cifs loaded successfully) until
> I edit->merge the 2nd ligand and then the bonds look wrong and when I try
> RSR it wants to force the ligand out and explodes the molecule.
>
> Original message: I thought that REFMAC tolerated dual occupancies if the
> sum of the two conformers was <= 1.0?
> Eleanor
> Will test..
>
> RE Pavel: I tried following that as a guide, however I’m running into an
> issue with restraints:
>
> I have made some progress troubleshooting the big cyclic molecule (ca6)
> and products (pA3). I re-editted the pdb; gave them the altlocs ABC; made
> sure they were same chain ID and residue number. However, when I try to
> real space refine with coot I get the error:
>
> “No restraints found! Non-existent or minimal description of restrained
> residues. Are you sure that you read a non-minimal mmCIF dictionary for
> this monomer? Are you sure the PDB residue name matches the dictionary
> residue name? If not, try File -> Import CIF dictionary. Alternatively, did
> you check that the atom names of the PDB file match those of the
> restraints? The residues in the chain are out of order. This can cause
> problems with residues selection. Suggest you re-order residues in
> increasing order.
>
> I definitely loaded the cifs. But it only allows me to rsr the ligand with
> altloc A. If cA6 is A then I can only rsr cA6. If pA3 is A then I can only
> refine pA3. When I delete->residue/monomer the cA6 with altloc A I can
> suddenly rsr the pA3, so both libraries must have been loaded successfully
> prior. I have included the snippet with the ligands just incase anyone
> wants to see. Funnily enough phenix.refmac allowed me to refine with no
> errors but it looks like it just ignored the pA3 as it did not properly fit
> itself into the density the way ca6 did.
>
> Original Message:
> Hi Stuart,
> the answer, I think, is here:
>
> https://phenix-online.org/phenixwebsite_static/mainsite/files/newsletter/CCN_2015_07.pdf#page=12
> Pavel
>
>
> RE George: I added allow_duplicate_sequence_numbers() to my
> coot-preferences but it didn’t seem to make a difference in my case. I
> could already open 1EJG with no error. My issue now appears to be the
> library file not recognising whichever ligand is not altloc A.
>
> Original Message:
> Dear Stuart,
> you have to add  allow_duplicate_sequence_numbers() to $HOME/.coot.py in
> OSX or the appropriate place on Windows. For
> Windows, as there is no $HOME, Coot uses .coot.py or .coot-preferences/
> directory for configuration - these can be found (added to) thedirectory in
> which Coot was installed (e.g. C:\WinCoot). Try with crambin (pdbcode
> 1EJG).This should give an error without allow_duplicate_sequence_numbers()
> but coot should start when added.
> Br, Georg.
>
> Final thoughts:
> I am now thinking that my other idea of splitting the ca6 into alternate
> conformers and then manually editing the pdb to make it into 2 product
> complexes with cyclised 2’-3’ phosphates on the ribose would also not work
> as the library would not be compatible.
>
> If anyone has any more ideas please let me know.
>
> Original query:
> I fit a large cyclic ligand cA6 (cylic hexa-adenylate) and after some
> refinement have noticed partial occupancy of it's hydrolysed form 2x A3,
> which has a cyclic 2'-3' phosphate on the terminal ribose.
>
> I tried fitting both ligands with 50% occupancy, but refinement doesn't
> allow them to occupy the same space.
>
> I subsequently tried text editing the pdb file, adding altloc identifiers
> to the ca6 and the 2 A3 molecules, making sure they were same chain and
> residue number as per this ccp4bb archive:
> http://www.phenix-online.org/pipermail/phenixbb/2011-March/016768.html.
> However, before I refined I checked in coot and all the ligand atoms are
> scattered and disconnected.
>
> I have thought about using coot to generate an alt conformation of ca6 and
> then text editing the B conformation to be split and have a 2'-3' cyclic
> phosphate. I am not sure if this is the correct way because it is a
> different chemical structure, so I could use 

Re: [ccp4bb] Refinement of ligand with alternate chemical structure

[Stuart McQuarrie]

Dear everyone that replied, thanks so much for the help thus far. 

Just for info I’m using Coot for Windows version 0.9.6 EL and phenix.refine 
version 1.20rc4-4416-000

RE Eleanor:  It appears my version of coot/refmac do not support separate 
ligands in the same space at <0.5 occupancy. As an experiment, I tried adding 
ATP and ADP at 0.45 occupancy to a different structure of mine. I’m able to 
real space refine in coot (so both .cifs loaded successfully) until I 
edit->merge the 2nd ligand and then the bonds look wrong and when I try RSR it 
wants to force the ligand out and explodes the molecule.

Original message: I thought that REFMAC tolerated dual occupancies if the sum 
of the two conformers was <= 1.0? 
Eleanor
Will test..

RE Pavel: I tried following that as a guide, however I’m running into an issue 
with restraints:

I have made some progress troubleshooting the big cyclic molecule (ca6) and 
products (pA3). I re-editted the pdb; gave them the altlocs ABC; made sure they 
were same chain ID and residue number. However, when I try to real space refine 
with coot I get the error: 

“No restraints found! Non-existent or minimal description of restrained 
residues. Are you sure that you read a non-minimal mmCIF dictionary for this 
monomer? Are you sure the PDB residue name matches the dictionary residue name? 
If not, try File -> Import CIF dictionary. Alternatively, did you check that 
the atom names of the PDB file match those of the restraints? The residues in 
the chain are out of order. This can cause problems with residues selection. 
Suggest you re-order residues in increasing order.

I definitely loaded the cifs. But it only allows me to rsr the ligand with 
altloc A. If cA6 is A then I can only rsr cA6. If pA3 is A then I can only 
refine pA3. When I delete->residue/monomer the cA6 with altloc A I can suddenly 
rsr the pA3, so both libraries must have been loaded successfully prior. I have 
included the snippet with the ligands just incase anyone wants to see. Funnily 
enough phenix.refmac allowed me to refine with no errors but it looks like it 
just ignored the pA3 as it did not properly fit itself into the density the way 
ca6 did. 

Original Message:
Hi Stuart,
the answer, I think, is here:
https://phenix-online.org/phenixwebsite_static/mainsite/files/newsletter/CCN_2015_07.pdf#page=12
Pavel


RE George: I added allow_duplicate_sequence_numbers() to my coot-preferences 
but it didn’t seem to make a difference in my case. I could already open 1EJG 
with no error. My issue now appears to be the library file not recognising 
whichever ligand is not altloc A. 

Original Message:
Dear Stuart, 
you have to add  allow_duplicate_sequence_numbers() to $HOME/.coot.py in OSX or 
the appropriate place on Windows. For
Windows, as there is no $HOME, Coot uses .coot.py or .coot-preferences/ 
directory for configuration - these can be found (added to) thedirectory in 
which Coot was installed (e.g. C:\WinCoot). Try with crambin (pdbcode 
1EJG).This should give an error without allow_duplicate_sequence_numbers() but 
coot should start when added.
Br, Georg.

Final thoughts:
I am now thinking that my other idea of splitting the ca6 into alternate 
conformers and then manually editing the pdb to make it into 2 product 
complexes with cyclised 2’-3’ phosphates on the ribose would also not work as 
the library would not be compatible. 

If anyone has any more ideas please let me know.  

Original query:
I fit a large cyclic ligand cA6 (cylic hexa-adenylate) and after some 
refinement have noticed partial occupancy of it's hydrolysed form 2x A3, which 
has a cyclic 2'-3' phosphate on the terminal ribose.

I tried fitting both ligands with 50% occupancy, but refinement doesn't allow 
them to occupy the same space.

I subsequently tried text editing the pdb file, adding altloc identifiers to 
the ca6 and the 2 A3 molecules, making sure they were same chain and residue 
number as per this ccp4bb archive: 
http://www.phenix-online.org/pipermail/phenixbb/2011-March/016768.html. 
However, before I refined I checked in coot and all the ligand atoms are 
scattered and disconnected.

I have thought about using coot to generate an alt conformation of ca6 and then 
text editing the B conformation to be split and have a 2'-3' cyclic phosphate. 
I am not sure if this is the correct way because it is a different chemical 
structure, so I could use some advice.

Kind regards,
Stuart


HETATM 5997  C1 AcA6 B 401  26.283  40.340  81.792  0.70 35.45   C  
HETATM 5998  C10AcA6 B 401  24.409  35.566  80.801  0.70 33.58   C  
HETATM 5999  C11AcA6 B 401  19.947  38.241  87.487  0.70 34.49   C  
HETATM 6000  C12AcA6 B 401  19.449  38.929  85.462  0.70 38.78   C  
HETATM 6001  C13AcA6 B 401  18.862  40.081  85.936  0.70 35.35   C  
HETATM 6002  C14AcA6 B 401  18.848  40.270  87.328  0.70 37.33   C  
HETATM 6003  C15AcA6 B 401  18.683  40.155  83.810  

Re: [ccp4bb] Refinement of ligand with alternate chemical structure

[Georg Mlynek]

Dear Stuart,

you have to add

  allow_duplicate_sequence_numbers()

to $HOME/.coot.py in OSX or the appropriate place on Windows. For
Windows, as there is no $HOME, Coot uses .coot.py or .coot-preferences/
directory for configuration - these can be found (added to) the
directory in which Coot was installed (e.g. C:\WinCoot).

Try with crambin (pdbcode 1EJG).This should give an error without 
allow_duplicate_sequence_numbers() but coot should start when added.


Br, Georg.


Am 15.02.2023 um 21:47 schrieb Nigel Moriarty:
One may need to set a preference in Coot to allow such a situation to 
be viewed. Paul helped me but I can't find the email.


Cheers

Nigel

---
Nigel W. Moriarty
Building 33R0349, Molecular Biophysics and Integrated Bioimaging
Lawrence Berkeley National Laboratory
Berkeley, CA 94720-8235
Email : nwmoria...@lbl.gov
Web  : CCI.LBL.gov 
ORCID : orcid.org/-0001-8857-9464 




On Wed, Feb 15, 2023 at 12:37 PM Eleanor Dodson 
<176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk> wrote:


I thought that REFMAC tolerated dual occupancies if the sum of the
two conformers was <= 1.0?
Eleanor
Will test..

On Wed, 15 Feb 2023 at 16:37, Stuart McQuarrie
<974c6ca32bc4-dmarc-requ...@jiscmail.ac.uk> wrote:

I fit a large cyclic ligand cA6 (cylic hexa-adenylate) and
after some refinement have noticed partial occupancy of it's
hydrolysed form 2x A3, which has a cyclic 2'-3' phosphate on
the terminal ribose.

I tried fitting both ligands with 50% occupancy, but
refinement doesn't allow them to occupy the same space.

I subsequently tried text editing the pdb file, adding altloc
identifiers to the ca6 and the 2 A3 molecules, making sure
they were same chain and residue number as per this ccp4bb
archive:
http://www.phenix-online.org/pipermail/phenixbb/2011-March/016768.html.
However, before I refined I checked in coot and all the ligand
atoms are scattered and disconnected.

I have thought about using coot to generate an alt
conformation of ca6 and then text editing the B conformation
to be split and have a 2'-3' cyclic phosphate. I am not sure
if this is the correct way because it is a different chemical
structure, so I could use some advice.

Kind regards,
Stuart



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Re: [ccp4bb] Refinement of ligand with alternate chemical structure

[Nigel Moriarty]

One may need to set a preference in Coot to allow such a situation to be
viewed. Paul helped me but I can't find the email.

Cheers

Nigel

---
Nigel W. Moriarty
Building 33R0349, Molecular Biophysics and Integrated Bioimaging
Lawrence Berkeley National Laboratory
Berkeley, CA 94720-8235
Email : nwmoria...@lbl.gov
Web  : CCI.LBL.gov
ORCID : orcid.org/-0001-8857-9464


On Wed, Feb 15, 2023 at 12:37 PM Eleanor Dodson <
176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk> wrote:

> I thought that REFMAC tolerated dual occupancies if the sum of the two
> conformers was <= 1.0?
> Eleanor
> Will test..
>
> On Wed, 15 Feb 2023 at 16:37, Stuart McQuarrie <
> 974c6ca32bc4-dmarc-requ...@jiscmail.ac.uk> wrote:
>
>> I fit a large cyclic ligand cA6 (cylic hexa-adenylate) and after some
>> refinement have noticed partial occupancy of it's hydrolysed form 2x A3,
>> which has a cyclic 2'-3' phosphate on the terminal ribose.
>>
>> I tried fitting both ligands with 50% occupancy, but refinement doesn't
>> allow them to occupy the same space.
>>
>> I subsequently tried text editing the pdb file, adding altloc identifiers
>> to the ca6 and the 2 A3 molecules, making sure they were same chain and
>> residue number as per this ccp4bb archive:
>> http://www.phenix-online.org/pipermail/phenixbb/2011-March/016768.html.
>> However, before I refined I checked in coot and all the ligand atoms are
>> scattered and disconnected.
>>
>> I have thought about using coot to generate an alt conformation of ca6
>> and then text editing the B conformation to be split and have a 2'-3'
>> cyclic phosphate. I am not sure if this is the correct way because it is a
>> different chemical structure, so I could use some advice.
>>
>> Kind regards,
>> Stuart
>>
>> 
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>>
>> This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a
>> mailing list hosted by www.jiscmail.ac.uk, terms & conditions are
>> available at https://www.jiscmail.ac.uk/policyandsecurity/
>>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
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>



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Re: [ccp4bb] Refinement of ligand with alternate chemical structure

[Eleanor Dodson]

I thought that REFMAC tolerated dual occupancies if the sum of the two
conformers was <= 1.0?
Eleanor
Will test..

On Wed, 15 Feb 2023 at 16:37, Stuart McQuarrie <
974c6ca32bc4-dmarc-requ...@jiscmail.ac.uk> wrote:

> I fit a large cyclic ligand cA6 (cylic hexa-adenylate) and after some
> refinement have noticed partial occupancy of it's hydrolysed form 2x A3,
> which has a cyclic 2'-3' phosphate on the terminal ribose.
>
> I tried fitting both ligands with 50% occupancy, but refinement doesn't
> allow them to occupy the same space.
>
> I subsequently tried text editing the pdb file, adding altloc identifiers
> to the ca6 and the 2 A3 molecules, making sure they were same chain and
> residue number as per this ccp4bb archive:
> http://www.phenix-online.org/pipermail/phenixbb/2011-March/016768.html.
> However, before I refined I checked in coot and all the ligand atoms are
> scattered and disconnected.
>
> I have thought about using coot to generate an alt conformation of ca6 and
> then text editing the B conformation to be split and have a 2'-3' cyclic
> phosphate. I am not sure if this is the correct way because it is a
> different chemical structure, so I could use some advice.
>
> Kind regards,
> Stuart
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>
> This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a
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Re: [ccp4bb] Refinement of ligand with alternate chemical structure

[Pavel Afonine]

Hi Stuart,
the answer, I think, is here:
https://phenix-online.org/phenixwebsite_static/mainsite/files/newsletter/CCN_2015_07.pdf#page=12
Pavel


On Wed, Feb 15, 2023 at 8:37 AM Stuart McQuarrie <
974c6ca32bc4-dmarc-requ...@jiscmail.ac.uk> wrote:

> I fit a large cyclic ligand cA6 (cylic hexa-adenylate) and after some
> refinement have noticed partial occupancy of it's hydrolysed form 2x A3,
> which has a cyclic 2'-3' phosphate on the terminal ribose.
>
> I tried fitting both ligands with 50% occupancy, but refinement doesn't
> allow them to occupy the same space.
>
> I subsequently tried text editing the pdb file, adding altloc identifiers
> to the ca6 and the 2 A3 molecules, making sure they were same chain and
> residue number as per this ccp4bb archive:
> http://www.phenix-online.org/pipermail/phenixbb/2011-March/016768.html.
> However, before I refined I checked in coot and all the ligand atoms are
> scattered and disconnected.
>
> I have thought about using coot to generate an alt conformation of ca6 and
> then text editing the B conformation to be split and have a 2'-3' cyclic
> phosphate. I am not sure if this is the correct way because it is a
> different chemical structure, so I could use some advice.
>
> Kind regards,
> Stuart
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>
> This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a
> mailing list hosted by www.jiscmail.ac.uk, terms & conditions are
> available at https://www.jiscmail.ac.uk/policyandsecurity/
>



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Re: [ccp4bb] refinement of 0.73A data in shelxl

[Tim Gruene]

Hi Phil,

yes, you are right. I mixed up the occupancy number with the part number.

I find these things easier in front of the res-file.

Thanks a lot for correcting this.

Best,
Tim

On Thursday, February 6, 2020 10:26:51 PM CET Phil Jeffrey wrote:
> That doesn't sound right re: PART numbers
> 
> classically:
> 
> PART 1
> majority disordered atoms with FVAR/occupancy of e.g. "21." instead
> of usual "11."
> PART 2
> minority disordered atoms with FVAR/occupancy of e.g. "-21."
> PART 0
> The 21.000/-21.000 pairs makes the sum of occupancies add to 1.0, but
> the actual value of each group is defined by the second free variable.
> 
> See: http://shelx.uni-goettingen.de/shelxl_html.php#PART
> The "PART 1" atoms would not interact with the "PART 2" atoms.
> There's even an example for a disordered SER in the documentation.
> 
> PART -n is used for disorders that overlap on themselves on symmetry
> axes.  "If n is negative, the generation of special position constraints
> is suppressed and bonds to symmetry generated atoms with the same or a
> different non-zero PART number are excluded; this is suitable for a
> solvent molecule disordered on a special position of higher symmetry
> than the molecule can take".
> 
> I use PART 1/PART 2/PART 0 all the time in "small molecule world" but
> I've used PART -1 precisely once.
> 
> Phil Jeffrey
> Princeton
> 
> On 2/6/20 4:15 PM, Tim Gruene wrote:
> > Dear Matthias,
> > 
> > 
> > some developers introduce new features of their refinement programs with
> > the words " ... which has been there in SHELXL since the beginning of
> > time".
> > 
> > If you are only looking for two conformations, you are looking for the
> > combination of free variable number N with part N and part -N. In case you
> > deal with more than two conformations, take a look at SUMP (as Jon
> > suggested).
> > 
> > The use of free variables is easier to explain right at the computer, so
> > please ask a colleague near you office, who is familiar with SHELXL for
> > the
> > details.
> > 
> > Best,
> > Tim
> > 
> > On Thursday, February 6, 2020 8:10:01 PM CET Barone, Matthias wrote:
> >> Sorry if the mail was not clear. I figured that out now yes. As I wrote
> >> in
> >> the update, I found this stupid error I made and now everything looks
> >> good.
> >> 
> >> Now that I got the feeling of how shelxl works, I miss one of it's
> >> features
> >> in the pdb format, namely the possibility to link occupancies of a double
> >> confirmation to another moiety, say a water or a double confirmation of
> >> the
> >> ligand. It's there a way to use something similar like FVAR in a pdb
> >> file?
> >> 
> >> 
> >> 
> >> 
> >> Dr. Matthias Barone
> >> 
> >> AG Kuehne, Rational Drug Design
> >> 
> >> Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
> >> Robert-Rössle-Strasse 10
> >> 13125 Berlin
> >> 
> >> Germany
> >> Phone: +49 (0)30 94793-284
> >> 
> >> 
> >> From: bogba...@yahoo.co.uk 
> >> Sent: Thursday, February 6, 2020 5:01:14 PM
> >> To: Barone, Matthias
> >> Cc: CCP4BB@JISCMAIL.AC.UK
> >> Subject: Re: [ccp4bb] refinement of 0.73A data in shelxl
> >> 
> >> 
> >> Hello, hope I can help.
> >> 
> >> 
> >> OK, so here is the disp table...
> >> 
> >> SFAC  C H CL N O
> >> 
> >> DISP $C 0.005100.00239 15.73708
> >> 
> >> DISP $H-0.20.0  0.66954
> >> 
> >> DISP $CL0.188450.21747   1035.16450
> >> 
> >> DISP $N 0.009540.00480 28.16118
> >> 
> >> DISP $O 0.016050.00875 47.79242
> >> 
> >> 
> >> If we take these coordinates...
> >> 
> >> N 30.414964   -0.1476350.11689611.00.19533
> >> 0.44341 =
> >> 
> >> H0A   20.427823   -0.1386560.12325611.0   -1.5
> >> 
> >> C 10.348035   -0.1607760.11097911.00.20723
> >> 0.28451 =
> >> 
> >> O 40.363785   -0.1741540.10290611.00.21226
> >> 0.22954 =
> >> 
> >> SG50.1773030.1012670.04057210.040000.06849
> >> 0.03024 =
> >> 
> >>

Re: [ccp4bb] refinement of 0.73A data in shelxl

[Phil Jeffrey]

That doesn't sound right re: PART numbers

classically:

PART 1
majority disordered atoms with FVAR/occupancy of e.g. "21." instead 
of usual "11."

PART 2
minority disordered atoms with FVAR/occupancy of e.g. "-21."
PART 0
The 21.000/-21.000 pairs makes the sum of occupancies add to 1.0, but 
the actual value of each group is defined by the second free variable.


See: http://shelx.uni-goettingen.de/shelxl_html.php#PART
The "PART 1" atoms would not interact with the "PART 2" atoms.
There's even an example for a disordered SER in the documentation.

PART -n is used for disorders that overlap on themselves on symmetry 
axes.  "If n is negative, the generation of special position constraints 
is suppressed and bonds to symmetry generated atoms with the same or a 
different non-zero PART number are excluded; this is suitable for a 
solvent molecule disordered on a special position of higher symmetry 
than the molecule can take".


I use PART 1/PART 2/PART 0 all the time in "small molecule world" but 
I've used PART -1 precisely once.


Phil Jeffrey
Princeton



On 2/6/20 4:15 PM, Tim Gruene wrote:

Dear Matthias,


some developers introduce new features of their refinement programs with the
words " ... which has been there in SHELXL since the beginning of time".

If you are only looking for two conformations, you are looking for the
combination of free variable number N with part N and part -N. In case you
deal with more than two conformations, take a look at SUMP (as Jon suggested).

The use of free variables is easier to explain right at the computer, so
please ask a colleague near you office, who is familiar with SHELXL for the
details.

Best,
Tim

On Thursday, February 6, 2020 8:10:01 PM CET Barone, Matthias wrote:

Sorry if the mail was not clear. I figured that out now yes. As I wrote in
the update, I found this stupid error I made and now everything looks good.

Now that I got the feeling of how shelxl works, I miss one of it's features
in the pdb format, namely the possibility to link occupancies of a double
confirmation to another moiety, say a water or a double confirmation of the
ligand. It's there a way to use something similar like FVAR in a pdb file?




Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: bogba...@yahoo.co.uk 
Sent: Thursday, February 6, 2020 5:01:14 PM
To: Barone, Matthias
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] refinement of 0.73A data in shelxl


Hello, hope I can help.


OK, so here is the disp table...

SFAC  C H CL N O

DISP $C 0.005100.00239 15.73708

DISP $H-0.20.0  0.66954

DISP $CL0.188450.21747   1035.16450

DISP $N 0.009540.00480 28.16118

DISP $O 0.016050.00875 47.79242


If we take these coordinates...

N 30.414964   -0.1476350.11689611.00.19533
0.44341 =

H0A   20.427823   -0.1386560.12325611.0   -1.5

C 10.348035   -0.1607760.11097911.00.20723
0.28451 =

O 40.363785   -0.1741540.10290611.00.21226
0.22954 =

SG50.1773030.1012670.04057210.040000.06849
0.03024 =

O 40.2413040.0717350.03856710.960000.14982
0.12755 =

... the first N (followed by 3) is being assigned the scattering factors of
chlorine because this element is 3rd in the SFAC list. The SG (followed by
5) is being assigned the scattering factors of O because the latter is 5th
in the SFAC list.

I think you need to check these  assignments and the chlorine occupancy are
Ok.

Jon Cooper

On 6 Feb 2020 11:13, "Barone, Matthias"  wrote:

Dear community
here is an update of my shelxl problem. I solved it after an epiphany last
night in bed... I tried countless things to get the postive density on the
Cl under control. Markus suggested that the density came from a radiolysed
chloride, so I tried to superimpose chlorinated and radiolysed ligands.
However that did not lead to anything fruitful.

Remember that I tried to incorporate DISP of Cl into the .ins file:
This is the original of the protein .ins, chloride just pasted as last
element: SFAC  C  H  N  O  S  CL
DISP $C 0.005100.00239 15.73708
DISP $H-0.20.0  0.66954
DISP $N 0.009540.00480 28.16118
DISP $O 0.016050.00875 47.79242
DISP $S 0.159950.16998812.87489
DISP $CL0.188450.21747   1035.16450

The upper list only creates postive density on the Chloride, the rest of the
map is clean and looks the same as if you would omit the DISP line of Cl
alltogether. The following list is coming from the .ins file of the
converted prodrg file:

SFAC  C H CL N O
DISP $C 0.005100.00239 15.73708
DISP 

Re: [ccp4bb] refinement of 0.73A data in shelxl

[Tim Gruene]

Dear Matthias,


some developers introduce new features of their refinement programs with the 
words " ... which has been there in SHELXL since the beginning of time".

If you are only looking for two conformations, you are looking for the 
combination of free variable number N with part N and part -N. In case you 
deal with more than two conformations, take a look at SUMP (as Jon suggested).

The use of free variables is easier to explain right at the computer, so 
please ask a colleague near you office, who is familiar with SHELXL for the 
details.

Best,
Tim

On Thursday, February 6, 2020 8:10:01 PM CET Barone, Matthias wrote:
> Sorry if the mail was not clear. I figured that out now yes. As I wrote in
> the update, I found this stupid error I made and now everything looks good.
> 
> Now that I got the feeling of how shelxl works, I miss one of it's features
> in the pdb format, namely the possibility to link occupancies of a double
> confirmation to another moiety, say a water or a double confirmation of the
> ligand. It's there a way to use something similar like FVAR in a pdb file?
> 
> 
> 
> 
> Dr. Matthias Barone
> 
> AG Kuehne, Rational Drug Design
> 
> Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
> Robert-Rössle-Strasse 10
> 13125 Berlin
> 
> Germany
> Phone: +49 (0)30 94793-284
> 
> 
> From: bogba...@yahoo.co.uk 
> Sent: Thursday, February 6, 2020 5:01:14 PM
> To: Barone, Matthias
> Cc: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] refinement of 0.73A data in shelxl
> 
> 
> Hello, hope I can help.
> 
> 
> OK, so here is the disp table...
> 
> SFAC  C H CL N O
> 
> DISP $C 0.005100.00239 15.73708
> 
> DISP $H-0.20.0  0.66954
> 
> DISP $CL0.188450.21747   1035.16450
> 
> DISP $N 0.009540.00480 28.16118
> 
> DISP $O 0.016050.00875 47.79242
> 
> 
> If we take these coordinates...
> 
> N 30.414964   -0.1476350.11689611.00.19533   
> 0.44341 =
> 
> H0A   20.427823   -0.1386560.12325611.0   -1.5
> 
> C 10.348035   -0.1607760.11097911.00.20723   
> 0.28451 =
> 
> O 40.363785   -0.1741540.10290611.00.21226   
> 0.22954 =
> 
> SG50.1773030.1012670.04057210.040000.06849   
> 0.03024 =
> 
> O 40.2413040.0717350.03856710.960000.14982   
> 0.12755 =
> 
> ... the first N (followed by 3) is being assigned the scattering factors of
> chlorine because this element is 3rd in the SFAC list. The SG (followed by
> 5) is being assigned the scattering factors of O because the latter is 5th
> in the SFAC list.
> 
> I think you need to check these  assignments and the chlorine occupancy are
> Ok.
> 
> Jon Cooper
> 
> On 6 Feb 2020 11:13, "Barone, Matthias"  wrote:
> 
> Dear community
> here is an update of my shelxl problem. I solved it after an epiphany last
> night in bed... I tried countless things to get the postive density on the
> Cl under control. Markus suggested that the density came from a radiolysed
> chloride, so I tried to superimpose chlorinated and radiolysed ligands.
> However that did not lead to anything fruitful.
> 
> Remember that I tried to incorporate DISP of Cl into the .ins file:
> This is the original of the protein .ins, chloride just pasted as last
> element: SFAC  C  H  N  O  S  CL
> DISP $C 0.005100.00239 15.73708
> DISP $H-0.20.0  0.66954
> DISP $N 0.009540.00480 28.16118
> DISP $O 0.016050.00875 47.79242
> DISP $S 0.159950.16998812.87489
> DISP $CL0.188450.21747   1035.16450
> 
> The upper list only creates postive density on the Chloride, the rest of the
> map is clean and looks the same as if you would omit the DISP line of Cl
> alltogether. The following list is coming from the .ins file of the
> converted prodrg file:
> 
> SFAC  C H CL N O
> DISP $C 0.005100.00239 15.73708
> DISP $H-0.20.0  0.66954
> DISP $CL0.188450.21747   1035.16450
> DISP $N 0.009540.00480 28.16118
> DISP $O 0.016050.00875 47.79242
> UNIT  38 48 1 5 7
> 
> Pasting CL as third element in the .ins file, however, created these weird
> difference signals on the backbone O and N that I mentioned. You can
> probably see where this is going. Here are some atoms of the protein in the
> .ins file:
> 
> N 30.414964   -0.1476350.11689611.00.19533   
> 0.44341 = H0A   20.427823   -0.1386560.12325611.0  
> -1.5 C 10.348

Re: [ccp4bb] refinement of 0.73A data in shelxl

[Mitchell D. Miller]

I am not sure about in the PDB format or mmCIF format.

However, other refinement programs have this ability too, e.g. the
occupancy groups of refmac
http://www.ysbl.york.ac.uk/refmac/data/refmac_keywords.html#Occupancy

buster refine has a tool pdb2occ to help generate appropriate  
groupings in gelly format

https://www.globalphasing.com/buster/wiki/index.cgi?AutoBusterLigandAndOccRef
http://www.globalphasing.com/pipermail/buster-discuss/2015-August/000255.html
which can be edited as needed. The recent release notes show this scripting
was improved recently -  
https://www.globalphasing.com/buster/ReleaseNotes/ReleaseNotes-BUSTER_snapshot_20200206.txt



and phenix.refine has occupancy groups as well, which are explained in  
detail in

this CCN newsletter article
https://www.phenix-online.org/newsletter/CCN_2015_07.pdf#page=12


Regards,
Mitch


Quoting "Barone, Matthias" :

Sorry if the mail was not clear. I figured that out now yes. As I  
wrote in the update, I found this stupid error I made and now  
everything looks good.


Now that I got the feeling of how shelxl works, I miss one of it's  
features in the pdb format, namely the possibility to link  
occupancies of a double confirmation to another moiety, say a water  
or a double confirmation of the ligand. It's there a way to use  
something similar like FVAR in a pdb file?





Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: bogba...@yahoo.co.uk 
Sent: Thursday, February 6, 2020 5:01:14 PM
To: Barone, Matthias
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] refinement of 0.73A data in shelxl


Hello, hope I can help.


OK, so here is the disp table...

SFAC  C H CL N O

DISP $C 0.005100.00239 15.73708

DISP $H-0.20.0  0.66954

DISP $CL0.188450.21747   1035.16450

DISP $N 0.009540.00480 28.16118

DISP $O 0.016050.00875 47.79242


If we take these coordinates...

N 30.414964   -0.1476350.11689611.00.19533
 0.44341 =


H0A   20.427823   -0.1386560.12325611.0   -1.5

C 10.348035   -0.1607760.11097911.00.20723
 0.28451 =


O 40.363785   -0.1741540.10290611.00.21226
 0.22954 =


SG50.1773030.1012670.04057210.040000.06849
 0.03024 =


O 40.2413040.0717350.03856710.960000.14982
 0.12755 =


... the first N (followed by 3) is being assigned the scattering  
factors of chlorine because this element is 3rd in the SFAC list.  
The SG (followed by 5) is being assigned the scattering factors of O  
because the latter is 5th in the SFAC list.


I think you need to check these  assignments and the chlorine  
occupancy are Ok.


Jon Cooper

On 6 Feb 2020 11:13, "Barone, Matthias"  wrote:

Dear community
here is an update of my shelxl problem. I solved it after an  
epiphany last night in bed...

I tried countless things to get the postive density on the Cl under control.
Markus suggested that the density came from a radiolysed chloride,  
so I tried to superimpose chlorinated and radiolysed ligands.

However that did not lead to anything fruitful.

Remember that I tried to incorporate DISP of Cl into the .ins file:
This is the original of the protein .ins, chloride just pasted as  
last element:

SFAC  C  H  N  O  S  CL
DISP $C 0.005100.00239 15.73708
DISP $H-0.20.0  0.66954
DISP $N 0.009540.00480 28.16118
DISP $O 0.016050.00875 47.79242
DISP $S 0.159950.16998812.87489
DISP $CL0.188450.21747   1035.16450

The upper list only creates postive density on the Chloride, the  
rest of the map is clean and looks the same as if you would omit the  
DISP line of Cl alltogether.

The following list is coming from the .ins file of the converted prodrg file:

SFAC  C H CL N O
DISP $C 0.005100.00239 15.73708
DISP $H-0.20.0  0.66954
DISP $CL0.188450.21747   1035.16450
DISP $N 0.009540.00480 28.16118
DISP $O 0.016050.00875 47.79242
UNIT  38 48 1 5 7

Pasting CL as third element in the .ins file, however, created these  
weird difference signals on the backbone O and N that I mentioned.
You can probably see where this is going. Here are some atoms of the  
protein in the .ins file:


N 30.414964   -0.1476350.11689611.00.19533
 0.44341 =

H0A   20.427823   -0.1386560.12325611.0   -1.5
C 10.348035   -0.1607760.11097911.00.20723
 0.28451 =
O 40.363785   -0.1741540.10290611.00.21226
 0.22954 =
SG50.1773030.1012670.04057210.040000.06849
 0.03024 =
O 40.2413040.0717350.038567   

Re: [ccp4bb] refinement of 0.73A data in shelxl

[00000c2488af9525-dmarc-request]

Hello, I think the secret of that type of coupled occupancy refinement lies in the SUMP command, but it's not one I have tangled with - others will know more.Jon CooperOn 6 Feb 2020 19:10, "Barone, Matthias"  wrote:


Sorry if the mail was not clear. I figured that out now yes. As I wrote in the update, I found this stupid error I made and now everything looks good. 
Now that I got the feeling of how shelxl works, I miss one of it's features in the pdb format, namely the possibility to link occupancies of a double confirmation to another moiety, say a water or a double confirmation of the ligand. It's there a way to
 use something similar like FVAR in a pdb file?








Dr. Matthias Barone
AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin
Germany
Phone: +49 (0)30 94793-284





From: bogbasic@yahoo.co.uk 
Sent: Thursday, February 6, 2020 5:01:14 PM
To: Barone, Matthias
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] refinement of 0.73A data in shelxl
 




Hello, hope I can help.


OK, so here is the disp table...

SFAC  C H CL N O  


DISP $C     0.00510    0.00239     15.73708


DISP $H    -0.2    0.0      0.66954


DISP $CL    0.18845    0.21747   1035.16450


DISP $N     0.00954    0.00480     28.16118


DISP $O     0.01605    0.00875     47.79242



If we take these coordinates...

N     3    0.414964   -0.147635    0.116896    11.0    0.19533    0.44341 =


H0A   2    0.427823   -0.138656    0.123256    11.0   -1.5


C     1    0.348035   -0.160776    0.110979    11.0    0.20723    0.28451 =


O     4    0.363785   -0.174154    0.102906    11.0    0.21226    0.22954 =


SG    5    0.177303    0.101267    0.040572    10.04000    0.06849    0.03024 =


O     4    0.241304    0.071735    0.038567    10.96000    0.14982    0.12755 =



... the first N (followed by 3) is being assigned the scattering factors of chlorine because this element is 3rd in the SFAC list. The SG (followed by 5) is being assigned the scattering factors of O because the latter is 5th in the SFAC list.
I think you need to check these  assignments and the chlorine occupancy are Ok.

Jon Cooper


On 6 Feb 2020 11:13, "Barone, Matthias"  wrote:





Dear community
here is an update of my shelxl problem. I solved it after an epiphany last night in bed...
I tried countless things to get the postive density on the Cl under control. 
Markus suggested that the density came from a radiolysed chloride, so I tried to superimpose chlorinated and radiolysed ligands.
However that did not lead to anything fruitful. 


Remember that I tried to incorporate DISP of Cl into the .ins file:
This is the original of the protein .ins, chloride just pasted as last element:
SFAC  C  H  N  O  S  CL
DISP $C     0.00510    0.00239     15.73708
DISP $H    -0.2    0.0      0.66954
DISP $N     0.00954    0.00480     28.16118
DISP $O     0.01605    0.00875     47.79242
DISP $S     0.15995    0.16998    812.87489
DISP $CL    0.18845    0.21747   1035.16450


The upper list only creates postive density on the Chloride, the rest of the map is clean and looks the same as if you would omit the DISP line of Cl alltogether.
The following list is coming from the .ins file of the converted prodrg file:


SFAC  C H CL N O  
DISP $C     0.00510    0.00239     15.73708
DISP $H    -0.2    0.0      0.66954
DISP $CL    0.18845    0.21747   1035.16450
DISP $N     0.00954    0.00480     28.16118
DISP $O     0.01605    0.00875     47.79242
UNIT  38 48 1 5 7  


Pasting CL as third element in the .ins file, however, created these weird difference signals on the backbone O and N that I mentioned. 
You can probably see where this is going. Here are some atoms of the protein in the .ins file:


N     3    0.414964   -0.147635    0.116896    11.0    0.19533    0.44341 =
H0A   2    0.427823   -0.138656    0.123256    11.0   -1.5
C     1    0.348035   -0.160776    0.110979    11.0    0.20723    0.28451 =
O     4    0.363785   -0.174154    0.102906    11.0    0.21226    0.22954 =
SG    5    0.177303    0.101267    0.040572    10.04000    0.06849    0.03024 =
O     4    0.241304    0.071735    0.038567    10.96000    0.14982    0.12755 =


And here are some atoms of the inhibitor:


OBM   5    0.325170    0.441790    0.181777    11.0    0.42576    0.30731 =  <- oxygen
CE1   1   -0.036497    0.262177    0.187030    11.0    0.12056    0.22455 =  <- carbon
HE1   2   -0.028898    0.247344    0.187663    11.0   -1.2
<- proton
NAY   4    0.107745    0.387704    0.210972    11.0    0.16719    0.14264 = <- nitrogen
CLAA   3  0.028744999  0.27121   0.199305996 0.5   
<- Chloride


Turned out that Jon had a good feeling about the swapping of

Re: [ccp4bb] refinement of 0.73A data in shelxl

[Barone, Matthias]

Sorry if the mail was not clear. I figured that out now yes. As I wrote in the 
update, I found this stupid error I made and now everything looks good.

Now that I got the feeling of how shelxl works, I miss one of it's features in 
the pdb format, namely the possibility to link occupancies of a double 
confirmation to another moiety, say a water or a double confirmation of the 
ligand. It's there a way to use something similar like FVAR in a pdb file?




Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: bogba...@yahoo.co.uk 
Sent: Thursday, February 6, 2020 5:01:14 PM
To: Barone, Matthias
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] refinement of 0.73A data in shelxl


Hello, hope I can help.


OK, so here is the disp table...

SFAC  C H CL N O

DISP $C 0.005100.00239 15.73708

DISP $H-0.20.0  0.66954

DISP $CL0.188450.21747   1035.16450

DISP $N 0.009540.00480 28.16118

DISP $O 0.016050.00875 47.79242


If we take these coordinates...

N 30.414964   -0.1476350.11689611.00.195330.44341 =

H0A   20.427823   -0.1386560.12325611.0   -1.5

C 10.348035   -0.1607760.11097911.00.207230.28451 =

O 40.363785   -0.1741540.10290611.00.212260.22954 =

SG50.1773030.1012670.04057210.040000.068490.03024 =

O 40.2413040.0717350.03856710.960000.149820.12755 =

... the first N (followed by 3) is being assigned the scattering factors of 
chlorine because this element is 3rd in the SFAC list. The SG (followed by 5) 
is being assigned the scattering factors of O because the latter is 5th in the 
SFAC list.

I think you need to check these  assignments and the chlorine occupancy are Ok.

Jon Cooper

On 6 Feb 2020 11:13, "Barone, Matthias"  wrote:

Dear community
here is an update of my shelxl problem. I solved it after an epiphany last 
night in bed...
I tried countless things to get the postive density on the Cl under control.
Markus suggested that the density came from a radiolysed chloride, so I tried 
to superimpose chlorinated and radiolysed ligands.
However that did not lead to anything fruitful.

Remember that I tried to incorporate DISP of Cl into the .ins file:
This is the original of the protein .ins, chloride just pasted as last element:
SFAC  C  H  N  O  S  CL
DISP $C 0.005100.00239 15.73708
DISP $H-0.20.0  0.66954
DISP $N 0.009540.00480 28.16118
DISP $O 0.016050.00875 47.79242
DISP $S 0.159950.16998812.87489
DISP $CL0.188450.21747   1035.16450

The upper list only creates postive density on the Chloride, the rest of the 
map is clean and looks the same as if you would omit the DISP line of Cl 
alltogether.
The following list is coming from the .ins file of the converted prodrg file:

SFAC  C H CL N O
DISP $C 0.005100.00239 15.73708
DISP $H-0.20.0  0.66954
DISP $CL0.188450.21747   1035.16450
DISP $N 0.009540.00480 28.16118
DISP $O 0.016050.00875 47.79242
UNIT  38 48 1 5 7

Pasting CL as third element in the .ins file, however, created these weird 
difference signals on the backbone O and N that I mentioned.
You can probably see where this is going. Here are some atoms of the protein in 
the .ins file:

N 30.414964   -0.1476350.11689611.00.195330.44341 =
H0A   20.427823   -0.1386560.12325611.0   -1.5
C 10.348035   -0.1607760.11097911.00.207230.28451 =
O 40.363785   -0.1741540.10290611.00.212260.22954 =
SG50.1773030.1012670.04057210.040000.068490.03024 =
O 40.2413040.0717350.03856710.960000.149820.12755 =

And here are some atoms of the inhibitor:

OBM   50.3251700.4417900.18177711.00.425760.30731 = 
 <- oxygen
CE1   1   -0.0364970.2621770.18703011.00.120560.22455 = 
 <- carbon
HE1   2   -0.0288980.2473440.18766311.0   -1.2 <- proton
NAY   40.1077450.3877040.21097211.00.167190.14264 = 
<- nitrogen
CLAA   3  0.028744999  0.27121   0.199305996 0.5<- Chloride

Turned out that Jon had a good feeling about the swapping of the lines and I 
did not understand Tim's comment "The scattering factor is derived from the 
number next to the name."
Once I adjusted the numbers in the second column of my inhibitors to match the 
DISP list numbering, Rfree dropped to 16.96% and the map looks notably better 
(see attached snap shot).


Again, thank you very much for such an incredibl

Re: [ccp4bb] refinement of 0.73A data in shelxl

[00000c2488af9525-dmarc-request]

Hello, hope I can help.OK, so here is the disp table...
SFAC  C H CL N O  
DISP $C     0.00510    0.00239     15.73708
DISP $H    -0.2    0.0      0.66954
DISP $CL    0.18845    0.21747   1035.16450
DISP $N     0.00954    0.00480     28.16118
DISP $O     0.01605    0.00875     47.79242If we take these coordinates...
N     3    0.414964   -0.147635    0.116896    11.0    0.19533    0.44341 =
H0A   2    0.427823   -0.138656    0.123256    11.0   -1.5
C     1    0.348035   -0.160776    0.110979    11.0    0.20723    0.28451 =
O     4    0.363785   -0.174154    0.102906    11.0    0.21226    0.22954 =
SG    5    0.177303    0.101267    0.040572    10.04000    0.06849    0.03024 =
O     4    0.241304    0.071735    0.038567    10.96000    0.14982    0.12755 =


... the first N (followed by 3) is being assigned the scattering factors of chlorine because this element is 3rd in the SFAC list. The SG (followed by 5) is being assigned the scattering factors of O because the latter is 5th in the SFAC list.
I think you need to check these  assignments and the chlorine occupancy are Ok.Jon CooperOn 6 Feb 2020 11:13, "Barone, Matthias"  wrote:





Dear community
here is an update of my shelxl problem. I solved it after an epiphany last night in bed...
I tried countless things to get the postive density on the Cl under control. 
Markus suggested that the density came from a radiolysed chloride, so I tried to superimpose chlorinated and radiolysed ligands.
However that did not lead to anything fruitful. 


Remember that I tried to incorporate DISP of Cl into the .ins file:
This is the original of the protein .ins, chloride just pasted as last element:
SFAC  C  H  N  O  S  CL
DISP $C     0.00510    0.00239     15.73708
DISP $H    -0.2    0.0      0.66954
DISP $N     0.00954    0.00480     28.16118
DISP $O     0.01605    0.00875     47.79242
DISP $S     0.15995    0.16998    812.87489
DISP $CL    0.18845    0.21747   1035.16450


The upper list only creates postive density on the Chloride, the rest of the map is clean and looks the same as if you would omit the DISP line of Cl alltogether.
The following list is coming from the .ins file of the converted prodrg file:


SFAC  C H CL N O  
DISP $C     0.00510    0.00239     15.73708
DISP $H    -0.2    0.0      0.66954
DISP $CL    0.18845    0.21747   1035.16450
DISP $N     0.00954    0.00480     28.16118
DISP $O     0.01605    0.00875     47.79242
UNIT  38 48 1 5 7  


Pasting CL as third element in the .ins file, however, created these weird difference signals on the backbone O and N that I mentioned. 
You can probably see where this is going. Here are some atoms of the protein in the .ins file:


N     3    0.414964   -0.147635    0.116896    11.0    0.19533    0.44341 =
H0A   2    0.427823   -0.138656    0.123256    11.0   -1.5
C     1    0.348035   -0.160776    0.110979    11.0    0.20723    0.28451 =
O     4    0.363785   -0.174154    0.102906    11.0    0.21226    0.22954 =
SG    5    0.177303    0.101267    0.040572    10.04000    0.06849    0.03024 =
O     4    0.241304    0.071735    0.038567    10.96000    0.14982    0.12755 =


And here are some atoms of the inhibitor:


OBM   5    0.325170    0.441790    0.181777    11.0    0.42576    0.30731 =  <- oxygen
CE1   1   -0.036497    0.262177    0.187030    11.0    0.12056    0.22455 =  <- carbon
HE1   2   -0.028898    0.247344    0.187663    11.0   -1.2
<- proton
NAY   4    0.107745    0.387704    0.210972    11.0    0.16719    0.14264 = <- nitrogen
CLAA   3  0.028744999  0.27121   0.199305996 0.5   
<- Chloride


Turned out that Jon had a good feeling about the swapping of the lines and I did not understand Tim's comment "The scattering factor is derived from the number next to the name."
Once I adjusted the numbers in the second column of my inhibitors to match the DISP list numbering, Rfree dropped to 16.96% and the map looks notably better (see attached snap shot).



Again, thank you very much for such an incredible feedback.
Best, Matthias










Dr. Matthias Barone
AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin
Germany
Phone: +49 (0)30 94793-284





From: CCP4 bulletin board  on behalf of Tim Gruene 
Sent: Tuesday, February 4, 2020 9:24:24 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] refinement of 0.73A data in shelxl
 



Dear Jon,

in SHELX(L), you can name your atoms foo and bar, or jon and doe, if you like. 
The scattering factor is derived from the number next to the name. The name is 
just that, and identifier.

Best,
Tim

On Monday, February 3, 2020 9:20:03 PM CET 0c2488af9525-dmarc-
request@JISCMAIL.AC.UK wrote:
> Remembered earlier that if the "CL"

Re: [ccp4bb] refinement of 0.73A data in shelxl

[Eleanor Dodson]

Dear Matias,
I fear some of your earlier problems are due to this: The atom types for
some of your ligand atoms, including the CL , are wrong in the PDB version
REFMAC and PHENIX both get the formfactor from the atom type..

Of course this does not matter to SHELX once it reformats the coordinates

ATOM 1874 CLAAAPRM A2000 1.044 16.706 17.649 1.13 9.18 * N*

ATOM 1875 NAYAPRM A2000 3.753 23.789 18.816 0.53 14.42 *O *

ATOM 1892 OALBPRM A2000 -1.518 19.920 21.181 0.32 25.06 *S*



Cheers Eleanor


On Thu, 6 Feb 2020 at 11:14, Barone, Matthias  wrote:

> Dear community
> here is an update of my shelxl problem. I solved it after an epiphany last
> night in bed...
> I tried countless things to get the postive density on the Cl under
> control.
> Markus suggested that the density came from a radiolysed chloride, so I
> tried to superimpose chlorinated and radiolysed ligands.
> However that did not lead to anything fruitful.
>
> Remember that I tried to incorporate DISP of Cl into the .ins file:
> This is the original of the protein .ins, chloride just pasted as last
> element:
> SFAC  C  H  N  O  S  CL
> DISP $C 0.005100.00239 15.73708
> DISP $H-0.20.0  0.66954
> DISP $N 0.009540.00480 28.16118
> DISP $O 0.016050.00875 47.79242
> DISP $S 0.159950.16998812.87489
> DISP $CL0.188450.21747   1035.16450
>
> The upper list only creates postive density on the Chloride, the rest of
> the map is clean and looks the same as if you would omit the DISP line of
> Cl alltogether.
> The following list is coming from the .ins file of the converted prodrg
> file:
>
> SFAC  C H CL N O
> DISP $C 0.005100.00239 15.73708
> DISP $H-0.20.0  0.66954
> DISP $CL0.188450.21747   1035.16450
> DISP $N 0.009540.00480 28.16118
> DISP $O 0.016050.00875 47.79242
> UNIT  38 48 1 5 7
>
> Pasting CL as third element in the .ins file, however, created these weird
> difference signals on the backbone O and N that I mentioned.
> You can probably see where this is going. Here are some atoms of the
> protein in the .ins file:
>
> N 30.414964   -0.1476350.11689611.00.19533
> 0.44341 =
> H0A   20.427823   -0.1386560.12325611.0   -1.5
> C 10.348035   -0.1607760.11097911.00.20723
> 0.28451 =
> O 40.363785   -0.1741540.10290611.00.21226
> 0.22954 =
> SG50.1773030.1012670.04057210.040000.06849
> 0.03024 =
> O 40.2413040.0717350.03856710.960000.14982
> 0.12755 =
>
> And here are some atoms of the inhibitor:
>
> OBM   50.3251700.4417900.18177711.00.42576
> 0.30731 =  <- oxygen
> CE1   1   -0.0364970.2621770.18703011.00.12056
> 0.22455 =  <- carbon
> HE1   2   -0.0288980.2473440.18766311.0   -1.2 <-
> proton
> NAY   40.1077450.3877040.21097211.00.16719
> 0.14264 = <- nitrogen
> CLAA   3  0.028744999  0.27121   0.199305996 0.5<-
> Chloride
>
> Turned out that Jon had a good feeling about the swapping of the lines and
> I did not understand Tim's comment "The scattering factor is derived from
> the number next to the name."
> Once I adjusted the numbers in the second column of my inhibitors to match
> the DISP list numbering, Rfree dropped to 16.96% and the map looks notably
> better (see attached snap shot).
>
>
> Again, thank you very much for such an incredible feedback.
>
> Best, Matthias
>
>
>
>
>
> Dr. Matthias Barone
>
> AG Kuehne, Rational Drug Design
>
> Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
> Robert-Rössle-Strasse 10
> 13125 Berlin
>
> Germany
> Phone: +49 (0)30 94793-284
> --
> *From:* CCP4 bulletin board  on behalf of Tim
> Gruene 
> *Sent:* Tuesday, February 4, 2020 9:24:24 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] refinement of 0.73A data in shelxl
>
> Dear Jon,
>
> in SHELX(L), you can name your atoms foo and bar, or jon and doe, if you
> like.
> The scattering factor is derived from the number next to the name. The
> name is
> just that, and identifier.
>
> Best,
> Tim
>
> On Monday, February 3, 2020 9:20:03 PM CET 0c2488af9525-dmarc-
> requ...@jiscmail.ac.uk wrote:
> > Remembered earlier that if the "CL" is not shifted one place to the left,
> > Shelx and probably most other programs treat it as carbon, i.e. its
> assumed
> > to have 6 rather than 17 electrons. Trust occupancies OK, too ;-?
> >
> >
> 

Re: [ccp4bb] refinement of 0.73A data in shelxl

[00000c2488af9525-dmarc-request]

Thanks, sorry, shelx is wonderful! I was thinking back to shelxpro days when your hetero-atoms could acquire the wrong scattering factors if they were not positioned right in the pdb file. The classic, which I saw several times, was calcium (CA) being treated as an alpha-carbon (CA), and I think refmac and others probably still do this if the PDB-standard leftward offset of the atom type is not present.Jon CooperOn 4 Feb 2020 08:24, Tim Gruene  wrote:Dear Jon,

in SHELX(L), you can name your atoms foo and bar, or jon and doe, if you like. 
The scattering factor is derived from the number next to the name. The name is 
just that, and identifier.

Best,
Tim

On Monday, February 3, 2020 9:20:03 PM CET 0c2488af9525-dmarc-
requ...@jiscmail.ac.uk wrote:
> Remembered earlier that if the "CL" is not shifted one place to the left,
> Shelx and probably most other programs treat it as carbon, i.e. its assumed
> to have 6 rather than 17 electrons. Trust occupancies OK, too ;-?
> 
> 
> Jon Cooper
> 
> 
> On 3 Feb 2020 18:26, "Barone, Matthias"  wrote:
> 
> 
> Hi Pavel
> 
> glad you write me. I was hoping you would read my post.
> 
> - Yes, protons are added, both on the protein as well as on the molecule
> 
> - I initially only refined protein and ligand anisotropically, now Im
> running a refinement with all atoms anisotrp except Hs. This would then
> also be the same as shelxl is doing.
> 
> - Alternate conformations are modeled, also on the ligand. There are plenty,
> sure, but I think I got most of them.
> 
> - I already used Water update during refine, there are some NO3s in the
> structure. I got them in. There is a second ligand somewhere as artifact.
> its density is not well defined, so I hope to get that in once the map
> clears up more. 
> 
> - I let phenix.refine optimize adp and chemisty weights, but as Petri
> suggested, Im manually increasing the scale factors to match the ones from
> shelxl (just to compare them properly). Im aiming for an rsmd of 0.02-0.03A
> like Petri suggested and keep an eye on how tight the structure is refined
> in shelxl.
> 
> 
> 
> 
> About the Rfact and the gap. Yes, thats what I was expecting. I hope if I
> add more anisotropic B fact, the Rfacts should go down to at least what
> shelxl yielded. 
> 
> 
> 
> 
> thank you all again for the massive feedback, ideas and help. 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> Dr. Matthias Barone
> 
> AG Kuehne, Rational Drug Design
> 
> 
> Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
> Robert-Rössle-Strasse 10
> 13125 Berlin
> 
> Germany
> Phone: +49 (0)30 94793-284
> 
> 
> From:Pavel Afonine 
> Sent:Monday, February 3, 2020 7:14:25 PM
> To:Barone, Matthias
> Cc:CCP4BB@JISCMAIL.AC.UK
> Subject:Re: [ccp4bb] refinement of 0.73A data in shelxl
>  
> Hi Matthias,
> 
> 
> did you use correct model parameterization and optimal refinement strategy
> for the resolution? Such as: - Add H atoms;
> - Refine all but H atoms with anisotropic ADPs;
> - Model alternative conformations (that one'd expect many at this
> resolution); - Add solvent (water, crystallization cocktail components if
> you see any); - Relax restraints on geometry and ADPs;
>  long list!
> 
> 
> If not, then what you have in terms of R factors is more or less what I'd
> expect.
> 
> 
> In the absence of obvious data pathologies, I'd expect Rwork/Rfree in 10-15%
> range, and the Rfree-Rwork gap around 1-2% or less.
> 
> 
> Since you mentioned Phenix refinement, I am happy to help you with details
> etc off-list.
> 
> 
> Pavel
> 
> 
> On Mon, Feb 3, 2020 at 3:08 AM Barone, Matthias 
> wrote:
> 
> 
> Dear ccp4 community
> 
> Im having some problems solving a 0.73A structure. Spacegroup seems to be
> correct, data are not twinned, 95.5% overall completeness, ISa 25.6. Outer
> shell CC1/2 24% and 90.4% complete.
> 
> The model is nearly fully built, there is no remaining unmodelled areas.
> However, Rfactisstuck 27% in phenix, with a very distinct artifact in the
> electron map (see phenix.jpg). You can see difference density on various
> well defined sidechain atoms. Notably, they seem to follow a pattern:
> Nearly all Val CG have difference signal, as well as many backbone
> NH. Hence, I suspected that it might be a problem with the SF, since we
> recorded the DS at 0.86A.
> 
> 
> 
> 
> Hence I gave shelxl a shot:
> 
> I used the refined model from phenix, converted it via pdb2ins and pasted
> the restraints created by prodrg. 
> 
> The shelxl hkl was produced by xdsconv, using the freeR flagging of the mtz
> used by phenix (no merge, friedel false).
> 
> Interestingly, shelxl can bring Rfree down to 16% and almost all of
> the diff-density artifacts seen before are gone (shelxl_noSFAC-CL.jpg).
> Except one: the inhibitor contains a chlorinated phenylring (pdb ligand
> 2L5) which now shows massive difference density for Cl.

Re: [ccp4bb] refinement of 0.73A data in shelxl

[Tim Gruene]

Dear Jon,

in SHELX(L), you can name your atoms foo and bar, or jon and doe, if you like. 
The scattering factor is derived from the number next to the name. The name is 
just that, and identifier.

Best,
Tim

On Monday, February 3, 2020 9:20:03 PM CET 0c2488af9525-dmarc-
requ...@jiscmail.ac.uk wrote:
> Remembered earlier that if the "CL" is not shifted one place to the left,
> Shelx and probably most other programs treat it as carbon, i.e. its assumed
> to have 6 rather than 17 electrons. Trust occupancies OK, too ;-?
> 
> 
> Jon Cooper
> 
> 
> On 3 Feb 2020 18:26, "Barone, Matthias"  wrote:
> 
> 
> Hi Pavel
> 
> glad you write me. I was hoping you would read my post.
> 
> - Yes, protons are added, both on the protein as well as on the molecule
> 
> - I initially only refined protein and ligand anisotropically, now Im
> running a refinement with all atoms anisotrp except Hs. This would then
> also be the same as shelxl is doing.
> 
> - Alternate conformations are modeled, also on the ligand. There are plenty,
> sure, but I think I got most of them.
> 
> - I already used Water update during refine, there are some NO3s in the
> structure. I got them in. There is a second ligand somewhere as artifact.
> its density is not well defined, so I hope to get that in once the map
> clears up more. 
> 
> - I let phenix.refine optimize adp and chemisty weights, but as Petri
> suggested, Im manually increasing the scale factors to match the ones from
> shelxl (just to compare them properly). Im aiming for an rsmd of 0.02-0.03A
> like Petri suggested and keep an eye on how tight the structure is refined
> in shelxl.
> 
> 
> 
> 
> About the Rfact and the gap. Yes, thats what I was expecting. I hope if I
> add more anisotropic B fact, the Rfacts should go down to at least what
> shelxl yielded. 
> 
> 
> 
> 
> thank you all again for the massive feedback, ideas and help. 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> Dr. Matthias Barone
> 
> AG Kuehne, Rational Drug Design
> 
> 
> Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
> Robert-Rössle-Strasse 10
> 13125 Berlin
> 
> Germany
> Phone: +49 (0)30 94793-284
> 
> 
> From:Pavel Afonine 
> Sent:Monday, February 3, 2020 7:14:25 PM
> To:Barone, Matthias
> Cc:CCP4BB@JISCMAIL.AC.UK
> Subject:Re: [ccp4bb] refinement of 0.73A data in shelxl
>  
> Hi Matthias,
> 
> 
> did you use correct model parameterization and optimal refinement strategy
> for the resolution? Such as: - Add H atoms;
> - Refine all but H atoms with anisotropic ADPs;
> - Model alternative conformations (that one'd expect many at this
> resolution); - Add solvent (water, crystallization cocktail components if
> you see any); - Relax restraints on geometry and ADPs;
>  long list!
> 
> 
> If not, then what you have in terms of R factors is more or less what I'd
> expect.
> 
> 
> In the absence of obvious data pathologies, I'd expect Rwork/Rfree in 10-15%
> range, and the Rfree-Rwork gap around 1-2% or less.
> 
> 
> Since you mentioned Phenix refinement, I am happy to help you with details
> etc off-list.
> 
> 
> Pavel
> 
> 
> On Mon, Feb 3, 2020 at 3:08 AM Barone, Matthias 
> wrote:
> 
> 
> Dear ccp4 community
> 
> Im having some problems solving a 0.73A structure. Spacegroup seems to be
> correct, data are not twinned, 95.5% overall completeness, ISa 25.6. Outer
> shell CC1/2 24% and 90.4% complete.
> 
> The model is nearly fully built, there is no remaining unmodelled areas.
> However, Rfactisstuck 27% in phenix, with a very distinct artifact in the
> electron map (see phenix.jpg). You can see difference density on various
> well defined sidechain atoms. Notably, they seem to follow a pattern:
> Nearly all Val CG have difference signal, as well as many backbone
> NH. Hence, I suspected that it might be a problem with the SF, since we
> recorded the DS at 0.86A.
> 
> 
> 
> 
> Hence I gave shelxl a shot:
> 
> I used the refined model from phenix, converted it via pdb2ins and pasted
> the restraints created by prodrg. 
> 
> The shelxl hkl was produced by xdsconv, using the freeR flagging of the mtz
> used by phenix (no merge, friedel false).
> 
> Interestingly, shelxl can bring Rfree down to 16% and almost all of
> the diff-density artifacts seen before are gone (shelxl_noSFAC-CL.jpg).
> Except one: the inhibitor contains a chlorinated phenylring (pdb ligand
> 2L5) which now shows massive difference density for Cl.
> 
> I therefore suggested that I might deal with a wrong SF for Cl. Funny
> enough, pdb2ins does not produce a DISP line for Cl if converting the pdb
> that contains the inhibitor. Hence, I used pdb2ins and the pdb from PRODRG
> to produce SFAC for the inhibitor Cloride. I then pasted this line
> 
> 
> DISP $CL0.188450.21747   1035.16450
> 
> 
> 
> into the .res file and updated the UNIT line. Shelxl runs through, and the
> density looks ok on the Chloride now. However Rfree is back up at 24% and
> the artifacts seen by phenix.refine are back (shelxl_SFAC-CL.jpg): now,
> very 

Re: [ccp4bb] refinement of 0.73A data in shelxl

[00000c2488af9525-dmarc-request]

Remembered earlier that if the "CL" is not shifted one place to the left, Shelx and probably most other programs treat it as carbon, i.e. its assumed to have 6 rather than 17 electrons. Trust occupancies OK, too ;-?Jon CooperOn 3 Feb 2020 18:26, "Barone, Matthias"  wrote:


Hi Pavel
glad you write me. I was hoping you would read my post.
- Yes, protons are added, both on the protein as well as on the molecule
- I initially only refined protein and ligand anisotropically, now Im running a refinement with all atoms anisotrp except Hs. This would then also be the same as shelxl is doing.
- Alternate conformations are modeled, also on the ligand. There are plenty, sure, but I think I got most of them.
- I already used Water update during refine, there are some NO3s in the structure. I got them in. There is a second ligand somewhere as artifact. its density is not well defined, so I hope to get that in once the map clears up more. 
- I let phenix.refine optimize adp and chemisty weights, but as Petri suggested, Im manually increasing the scale factors to match the ones from shelxl (just to compare them properly). Im aiming for an rsmd of 0.02-0.03A like Petri suggested and keep an
 eye on how tight the structure is refined in shelxl.


About the Rfact and the gap. Yes, thats what I was expecting. I hope if I add more anisotropic B fact, the Rfacts should go down to at least what shelxl yielded. 


thank you all again for the massive feedback, ideas and help. 








Dr. Matthias Barone
AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin
Germany
Phone: +49 (0)30 94793-284





From: Pavel Afonine 
Sent: Monday, February 3, 2020 7:14:25 PM
To: Barone, Matthias
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] refinement of 0.73A data in shelxl
 



Hi Matthias,


did you use correct model parameterization and optimal refinement strategy for the resolution? Such as:
- Add H atoms;
- Refine all but H atoms with anisotropic ADPs;
- Model alternative conformations (that one'd expect many at this resolution);
- Add solvent (water, crystallization cocktail components if you see any);
- Relax restraints on geometry and ADPs;
 long list!


If not, then what you have in terms of R factors is more or less what I'd expect.


In the absence of obvious data pathologies, I'd expect Rwork/Rfree in 10-15% range, and the Rfree-Rwork gap around 1-2% or less.


Since you mentioned Phenix refinement, I am happy to help you with details etc off-list.


Pavel


On Mon, Feb 3, 2020 at 3:08 AM Barone, Matthias <Barone@fmp-berlin.de> wrote:




Dear ccp4 community
Im having some problems solving a 0.73A structure. Spacegroup seems to be correct,
 data are not twinned, 95.5% overall completeness, ISa 25.6. Outer shell CC1/2
 24% and 90.4% complete.
The model is nearly fully built, there is no remaining unmodelled areas. However, Rfact
is stuck 27% in phenix, with a very distinct artifact in the electron map (see phenix.jpg). You can see difference
 density on various well defined sidechain atoms. Notably, they seem to follow a pattern: Nearly all Val CG have difference signal, as well as many backbone NH. Hence, I suspected that it might be a problem with the SF, since
 we recorded the DS at 0.86A. 



Hence I gave shelxl a shot:
I used the refined model from phenix, converted it via pdb2ins and pasted the restraints created by prodrg. 
The shelxl hkl was produced by xdsconv, using the freeR flagging of the mtz used by phenix (no merge, friedel false).
Interestingly, shelxl can bring Rfree down to 16% and almost all of the diff-density artifacts seen before are gone (shelxl_noSFAC-CL.jpg). Except one: the inhibitor contains a chlorinated phenylring (pdb
 ligand 2L5) which now shows massive difference density for Cl. 
I therefore suggested that I might deal with a wrong SF for Cl. Funny enough, pdb2ins does not produce a DISP line for Cl if converting the pdb that contains the inhibitor. Hence,
I used pdb2ins and the pdb from PRODRG to produce SFAC for the inhibitor Cloride. I then pasted this line

DISP $CL    0.18845    0.21747   1035.16450

into the .res file and updated the UNIT line. Shelxl runs through, and the density looks ok on the Chloride now. However Rfree is back
 up at 24% and the artifacts seen by phenix.refine are back (shelxl_SFAC-CL.jpg): now, very distincitvly, backbone carbonyls and NHs show difference
 density.

Am I right in my assumption, that the SFAC of Cloride is not properly calculated at the given wavelenght? And if so, how do I guess it correctly?


Thank you very much for your help!
Best, matthias








Dr. Matthias Barone
AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin
Germany

Re: [ccp4bb] refinement of 0.73A data in shelxl

[Barone, Matthias]

Hi Pavel

glad you write me. I was hoping you would read my post.

- Yes, protons are added, both on the protein as well as on the molecule

- I initially only refined protein and ligand anisotropically, now Im running a 
refinement with all atoms anisotrp except Hs. This would then also be the same 
as shelxl is doing.

- Alternate conformations are modeled, also on the ligand. There are plenty, 
sure, but I think I got most of them.

- I already used Water update during refine, there are some NO3s in the 
structure. I got them in. There is a second ligand somewhere as artifact. its 
density is not well defined, so I hope to get that in once the map clears up 
more.

- I let phenix.refine optimize adp and chemisty weights, but as Petri 
suggested, Im manually increasing the scale factors to match the ones from 
shelxl (just to compare them properly). Im aiming for an rsmd of 0.02-0.03A 
like Petri suggested and keep an eye on how tight the structure is refined in 
shelxl.


About the Rfact and the gap. Yes, thats what I was expecting. I hope if I add 
more anisotropic B fact, the Rfacts should go down to at least what shelxl 
yielded.


thank you all again for the massive feedback, ideas and help.




Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284


From: Pavel Afonine 
Sent: Monday, February 3, 2020 7:14:25 PM
To: Barone, Matthias
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] refinement of 0.73A data in shelxl

Hi Matthias,

did you use correct model parameterization and optimal refinement strategy for 
the resolution? Such as:
- Add H atoms;
- Refine all but H atoms with anisotropic ADPs;
- Model alternative conformations (that one'd expect many at this resolution);
- Add solvent (water, crystallization cocktail components if you see any);
- Relax restraints on geometry and ADPs;
 long list!

If not, then what you have in terms of R factors is more or less what I'd 
expect.

In the absence of obvious data pathologies, I'd expect Rwork/Rfree in 10-15% 
range, and the Rfree-Rwork gap around 1-2% or less.

Since you mentioned Phenix refinement, I am happy to help you with details etc 
off-list.

Pavel

On Mon, Feb 3, 2020 at 3:08 AM Barone, Matthias 
mailto:bar...@fmp-berlin.de>> wrote:

Dear ccp4 community

Im having some problems solving a 0.73A structure. Spacegroup seems to be 
correct, data are not twinned, 95.5% overall completeness, ISa 25.6. Outer 
shell CC1/2 24% and 90.4% complete.

The model is nearly fully built, there is no remaining unmodelled areas. 
However, Rfact is stuck 27% in phenix, with a very distinct artifact in the 
electron map (see phenix.jpg). You can see difference density on various well 
defined sidechain atoms. Notably, they seem to follow a pattern: Nearly all Val 
CG have difference signal, as well as many backbone NH. Hence, I suspected that 
it might be a problem with the SF, since we recorded the DS at 0.86A.


Hence I gave shelxl a shot:

I used the refined model from phenix, converted it via pdb2ins and pasted the 
restraints created by prodrg.

The shelxl hkl was produced by xdsconv, using the freeR flagging of the mtz 
used by phenix (no merge, friedel false).

Interestingly, shelxl can bring Rfree down to 16% and almost all of the 
diff-density artifacts seen before are gone (shelxl_noSFAC-CL.jpg). Except one: 
the inhibitor contains a chlorinated phenylring (pdb ligand 2L5) which now 
shows massive difference density for Cl.

I therefore suggested that I might deal with a wrong SF for Cl. Funny enough, 
pdb2ins does not produce a DISP line for Cl if converting the pdb that contains 
the inhibitor. Hence, I used pdb2ins and the pdb from PRODRG to produce SFAC 
for the inhibitor Cloride. I then pasted this line

DISP $CL0.188450.21747   1035.16450

into the .res file and updated the UNIT line. Shelxl runs through, and the 
density looks ok on the Chloride now. However Rfree is back up at 24% and the 
artifacts seen by phenix.refine are back (shelxl_SFAC-CL.jpg): now, very 
distincitvly, backbone carbonyls and NHs show difference density.

Am I right in my assumption, that the SFAC of Cloride is not properly 
calculated at the given wavelenght? And if so, how do I guess it correctly?


Thank you very much for your help!

Best, matthias




Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284



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Re: [ccp4bb] refinement of 0.73A data in shelxl

[Pavel Afonine]

Hi Matthias,

did you use correct model parameterization and optimal refinement strategy
for the resolution? Such as:
- Add H atoms;
- Refine all but H atoms with anisotropic ADPs;
- Model alternative conformations (that one'd expect many at this
resolution);
- Add solvent (water, crystallization cocktail components if you see any);
- Relax restraints on geometry and ADPs;
 long list!

If not, then what you have in terms of R factors is more or less what I'd
expect.

In the absence of obvious data pathologies, I'd expect Rwork/Rfree in
10-15% range, and the Rfree-Rwork gap around 1-2% or less.

Since you mentioned Phenix refinement, I am happy to help you with details
etc off-list.

Pavel

On Mon, Feb 3, 2020 at 3:08 AM Barone, Matthias 
wrote:

> Dear ccp4 community
>
> Im having some problems solving a 0.73A structure. Spacegroup seems to be
> correct, data are not twinned, 95.5% overall completeness, ISa 25.6. Outer
> shell CC1/2 24% and 90.4% complete.
>
> The model is nearly fully built, there is no remaining unmodelled areas.
> However, Rfact is stuck 27% in phenix, with a very distinct artifact in
> the electron map (see phenix.jpg). You can see difference density on
> various well defined sidechain atoms. Notably, they seem to follow a
> pattern: Nearly all Val CG have difference signal, as well as many backbone
> NH. Hence, I suspected that it might be a problem with the SF, since we
> recorded the DS at 0.86A.
>
>
> Hence I gave shelxl a shot:
>
> I used the refined model from phenix, converted it via pdb2ins and pasted
> the restraints created by prodrg.
>
> The shelxl hkl was produced by xdsconv, using the freeR flagging of the
> mtz used by phenix (no merge, friedel false).
>
> Interestingly, shelxl can bring Rfree down to 16% and almost all of
> the diff-density artifacts seen before are gone (shelxl_noSFAC-CL.jpg).
> Except one: the inhibitor contains a chlorinated phenylring (pdb ligand
> 2L5) which now shows massive difference density for Cl.
>
> I therefore suggested that I might deal with a wrong SF for Cl. Funny
> enough, pdb2ins does not produce a DISP line for Cl if converting the pdb
> that contains the inhibitor. Hence, I used pdb2ins and the pdb from
> PRODRG to produce SFAC for the inhibitor Cloride. I then pasted this line
>
> DISP $CL0.188450.21747   1035.16450
>
> into the .res file and updated the UNIT line. Shelxl runs through, and
> the density looks ok on the Chloride now. However Rfree is back up at 24%
> and the artifacts seen by phenix.refine are back (shelxl_SFAC-CL.jpg):
> now, very distincitvly, backbone carbonyls and NHs show difference density.
>
> Am I right in my assumption, that the SFAC of Cloride is not properly
> calculated at the given wavelenght? And if so, how do I guess it correctly?
>
>
> Thank you very much for your help!
>
> Best, matthias
>
>
>
>
> Dr. Matthias Barone
>
> AG Kuehne, Rational Drug Design
>
> Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
> Robert-Rössle-Strasse 10
> 13125 Berlin
>
> Germany
> Phone: +49 (0)30 94793-284
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] refinement of 0.73A data in shelxl

[00000c2488af9525-dmarc-request]

Hello, sorry if this is a really obvious question, but have you used the ANIS option? I remember it is good at cleaning-up difference density around halogen atoms that sort of resolution.Jon CooperOn 3 Feb 2020 11:08, "Barone, Matthias"  wrote:

Dear ccp4 community
Im having some problems solving a 0.73A structure. Spacegroup
 seems to be correct, data are not twinned, 95.5% overall completeness, ISa 25.6. Outer shell CC1/2
 24% and 90.4% complete.
The model is nearly fully built, there is no remaining unmodelled
 areas. However, Rfact is stuck 27% in phenix, with a very distinct artifact in the electron map (see phenix.jpg).
 You can see difference density on various well defined sidechain atoms. Notably, they seem to follow a pattern: Nearly all Val CG have difference signal, as well as many backbone NH. Hence, I suspected that it might be
 a problem with the SF, since we recorded the DS at 0.86A. 



Hence I gave shelxl a shot:
I used the refined model from phenix, converted it via pdb2ins and pasted the restraints created by prodrg. 
The shelxl hkl was produced by xdsconv, using the freeR flagging of the mtz used by phenix (no merge, friedel false).
Interestingly, shelxl can bring Rfree down to 16% and almost all of the diff-density artifacts seen before are gone (shelxl_noSFAC-CL.jpg). Except one: the inhibitor contains a chlorinated phenylring
 (pdb ligand 2L5) which now shows massive difference density for Cl. 
I therefore suggested that I might deal with a wrong SF for Cl. Funny enough, pdb2ins does not produce a DISP line for Cl if converting the pdb that contains the inhibitor. Hence,
I used pdb2ins and the pdb from PRODRG to produce SFAC for the inhibitor Cloride. I then pasted this line

DISP $CL    0.18845    0.21747   1035.16450

into the .res file and updated the UNIT line. Shelxl runs through, and the density looks ok on the Chloride now. However Rfree
 is back up at 24% and the artifacts seen by phenix.refine are back (shelxl_SFAC-CL.jpg): now, very distincitvly, backbone carbonyls and
 NHs show difference density.

Am I right in my assumption, that the SFAC of Cloride is not properly calculated at the given wavelenght? And if so, how do I guess it correctly?


Thank you very much for your help!
Best, matthias








Dr. Matthias Barone
AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin
Germany
Phone: +49 (0)30 94793-284







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Re: [ccp4bb] refinement of 0.73A data in shelxl

[Bernhard Rupp]

Maybe the figures were just contoured odd...but my recollection of a good
0.8 A map appearance is differentwhich might be consistent with the
high Rf...a look at the high res data or images/stats might help?

Best, br

On Mon, Feb 3, 2020, 14:58 George Sheldrick 
wrote:

> Dear Matthias,
>
>
> That is very strange. First please repeat the shelxl refinement with the
> occupancy of the offending chlorine(s) in the .ins file changed from 11
> (i.e. fixed at 1.0) to 1.0 (i.e. freely refined starting from 1.0). If that
> does not help I would be happy to look at it in confidence, I would need
> the .hkl and .ins files.
>
>
> Best wishes, George
>
>
>
>
> On 03.02.20 12:08, Barone, Matthias wrote:
>
> Dear ccp4 community
>
> Im having some problems solving a 0.73A structure. Spacegroup seems to be
> correct, data are not twinned, 95.5% overall completeness, ISa 25.6. Outer
> shell CC1/2 24% and 90.4% complete.
>
> The model is nearly fully built, there is no remaining unmodelled areas.
> However, Rfact is stuck 27% in phenix, with a very distinct artifact in
> the electron map (see phenix.jpg). You can see difference density on
> various well defined sidechain atoms. Notably, they seem to follow a
> pattern: Nearly all Val CG have difference signal, as well as many backbone
> NH. Hence, I suspected that it might be a problem with the SF, since we
> recorded the DS at 0.86A.
>
>
> Hence I gave shelxl a shot:
>
> I used the refined model from phenix, converted it via pdb2ins and pasted
> the restraints created by prodrg.
>
> The shelxl hkl was produced by xdsconv, using the freeR flagging of the
> mtz used by phenix (no merge, friedel false).
>
> Interestingly, shelxl can bring Rfree down to 16% and almost all of
> the diff-density artifacts seen before are gone (shelxl_noSFAC-CL.jpg).
> Except one: the inhibitor contains a chlorinated phenylring (pdb ligand
> 2L5) which now shows massive difference density for Cl.
>
> I therefore suggested that I might deal with a wrong SF for Cl. Funny
> enough, pdb2ins does not produce a DISP line for Cl if converting the pdb
> that contains the inhibitor. Hence, I used pdb2ins and the pdb from
> PRODRG to produce SFAC for the inhibitor Cloride. I then pasted this line
>
> DISP $CL0.188450.21747   1035.16450
>
> into the .res file and updated the UNIT line. Shelxl runs through, and
> the density looks ok on the Chloride now. However Rfree is back up at 24%
> and the artifacts seen by phenix.refine are back (shelxl_SFAC-CL.jpg):
> now, very distincitvly, backbone carbonyls and NHs show difference density.
>
> Am I right in my assumption, that the SFAC of Cloride is not properly
> calculated at the given wavelenght? And if so, how do I guess it correctly?
>
> Thank you very much for your help!
>
> Best, matthias
>
>
> Dr. Matthias Barone
>
> AG Kuehne, Rational Drug Design
>
> Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
> Robert-Rössle-Strasse 10
> 13125 Berlin
>
> Germany
> Phone: +49 (0)30 94793-284
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>
> --
> Prof. George M. Sheldrick FRS
> Dept. Structural Chemistry
> University of Goettingen
> Tammannstr.  4
> D37077 Goettingen
> Germany
> Tel: +49 551 3933021 or +49 5594 227312
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] refinement of 0.73A data in shelxl

[George Sheldrick]

Dear Matthias,


That is very strange. First please repeat the shelxl refinement with the 
occupancy of the offending chlorine(s) in the .ins file changed from 11  
(i.e. fixed at 1.0) to 1.0 (i.e. freely refined starting from 1.0). If 
that does not help I would be happy to look at it in confidence, I would 
need the .hkl and .ins files.



Best wishes, George




On 03.02.20 12:08, Barone, Matthias wrote:


Dear ccp4 community

Im having some problems solving a 0.73A structure. Spacegroup seems to 
be correct, data are not twinned, 95.5% overall completeness, ISa 
25.6. Outer shell CC1/2 24% and 90.4% complete.


The model is nearly fully built, there is no remaining unmodelled 
areas. However, Rfact isstuck 27% in phenix, with a very distinct 
artifact in the electron map (see phenix.jpg). You can see difference 
density on various well defined sidechain atoms. Notably, they seem to 
follow a pattern: Nearly all Val CG have difference signal, as well as 
many backbone NH. Hence, I suspected that it might be a problem with 
the SF, since we recorded the DS at 0.86A.



Hence I gave shelxl a shot:

I used the refined model from phenix, converted it via pdb2ins and 
pasted the restraints created by prodrg.


The shelxl hkl was produced by xdsconv, using the freeR flagging of 
the mtz used by phenix (no merge, friedel false).


Interestingly, shelxl can bring Rfree down to 16% and almost all of 
the diff-density artifacts seen before are 
gone (shelxl_noSFAC-CL.jpg). Except one: the inhibitor contains a 
chlorinated phenylring (pdb ligand 2L5) which now shows massive 
difference density for Cl.


I therefore suggested that I might deal with a wrong SF for Cl. Funny 
enough, pdb2ins does not produce a DISP line for Cl if converting the 
pdb that contains the inhibitor. Hence, I used pdb2ins and the pdb 
from PRODRG to produce SFAC for the inhibitor Cloride. I then pasted 
this line


DISP $CL    0.18845    0.21747   1035.16450

into the .res file andupdated the UNIT line. Shelxl runs through, and 
the density looks ok on the Chloride now. However Rfree is back up at 
24% and the artifacts seen by phenix.refine are back 
(shelxl_SFAC-CL.jpg): now, very distincitvly, backbone carbonyls and 
NHs show difference density.


Am I right in my assumption, that the SFAC of Cloride is not properly 
calculated at the given wavelenght? And if so, how do I guess it 
correctly?


Thank you very much for your help!

Best, matthias


Dr. Matthias Barone

AG Kuehne, Rational Drug Design

Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
Robert-Rössle-Strasse 10
13125 Berlin

Germany
Phone: +49 (0)30 94793-284




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--
Prof. George M. Sheldrick FRS
Dept. Structural Chemistry
University of Goettingen
Tammannstr.  4
D37077 Goettingen
Germany
Tel: +49 551 3933021 or +49 5594 227312




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Re: [ccp4bb] refinement of 0.73A data in shelxl

[Eleanor Dodson]

The maps look beautiful, but maybe the high resolution data is refining
poorly.
Not sure how to do this with phenix or SHELX but REFMAC gives you a plot of
 and  v resolution. You sometimes see wild divergence in some
resolution shells.
Looking at Rfactors v resolution can also highlight such problems.

Eleanor


On Mon, 3 Feb 2020 at 11:08, Barone, Matthias  wrote:

> Dear ccp4 community
>
> Im having some problems solving a 0.73A structure. Spacegroup seems to be
> correct, data are not twinned, 95.5% overall completeness, ISa 25.6. Outer
> shell CC1/2 24% and 90.4% complete.
>
> The model is nearly fully built, there is no remaining unmodelled areas.
> However, Rfact is stuck 27% in phenix, with a very distinct artifact in
> the electron map (see phenix.jpg). You can see difference density on
> various well defined sidechain atoms. Notably, they seem to follow a
> pattern: Nearly all Val CG have difference signal, as well as many backbone
> NH. Hence, I suspected that it might be a problem with the SF, since we
> recorded the DS at 0.86A.
>
>
> Hence I gave shelxl a shot:
>
> I used the refined model from phenix, converted it via pdb2ins and pasted
> the restraints created by prodrg.
>
> The shelxl hkl was produced by xdsconv, using the freeR flagging of the
> mtz used by phenix (no merge, friedel false).
>
> Interestingly, shelxl can bring Rfree down to 16% and almost all of
> the diff-density artifacts seen before are gone (shelxl_noSFAC-CL.jpg).
> Except one: the inhibitor contains a chlorinated phenylring (pdb ligand
> 2L5) which now shows massive difference density for Cl.
>
> I therefore suggested that I might deal with a wrong SF for Cl. Funny
> enough, pdb2ins does not produce a DISP line for Cl if converting the pdb
> that contains the inhibitor. Hence, I used pdb2ins and the pdb from
> PRODRG to produce SFAC for the inhibitor Cloride. I then pasted this line
>
> DISP $CL0.188450.21747   1035.16450
>
> into the .res file and updated the UNIT line. Shelxl runs through, and
> the density looks ok on the Chloride now. However Rfree is back up at 24%
> and the artifacts seen by phenix.refine are back (shelxl_SFAC-CL.jpg):
> now, very distincitvly, backbone carbonyls and NHs show difference density.
>
> Am I right in my assumption, that the SFAC of Cloride is not properly
> calculated at the given wavelenght? And if so, how do I guess it correctly?
>
>
> Thank you very much for your help!
>
> Best, matthias
>
>
>
>
> Dr. Matthias Barone
>
> AG Kuehne, Rational Drug Design
>
> Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
> Robert-Rössle-Strasse 10
> 13125 Berlin
>
> Germany
> Phone: +49 (0)30 94793-284
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] Refinement of Occupancy in Refmac

[Christian Roth]

Hi Deniz,
yes you can do that, though I don't know right now how B-factors are taken
into account when refining occupancies.
Anyway, in i2 you have to go to the advanced tab. There is a  place to put
keywords in (text area). There you put the right combination of keywords
for your special cases. Regarding syntax, please look here.

http://www.ysbl.york.ac.uk/refmac/data/refmac_keywords.html#Occupancy

Hope you find the right combination of keywords and it will work out for you

Cheers

Christian

On Mon, Jun 24, 2019 at 10:57 PM "Deniz Üresin" 
wrote:

> Hello,
> when I add an alternative conformation of a residue in COOT, I have to set
> the occupancy of both conformers, but Refmac5 won't touch these numbers.
> Can I tell Refmac to check for the best fitting occupancies (in the Windows
> version of CCP4i2)?
>
> With kind regards,
> Deniz Üresin
>
> Institute for Biophysics
> Goethe-University Frankfurt
> Max-von-Laue-Straße 1
> 60438 Frankfurt/Main
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] Refinement

[Raymond Brown]

Hi,

Run your data through POINTLESS to check space group.

Ray

On Sun, 3/24/19, StrBio  wrote:

 Subject: [ccp4bb] Refinement
 To: CCP4BB@JISCMAIL.AC.UK
 Date: Sunday, March 24, 2019, 12:17 AM
 
 ALL.
 I have data at 2.4 A in P21 sp gr, helical
 protein. 
 Refined to Rwork 29 Rfree 34 with nice density
 map and all nice statistics oither Rfactor (by Phenix).
 Refmac quit same. 
 Should I deposit it or look better
 data?Any suggestion?
   
 
 
 
 
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Re: [ccp4bb] Refinement

[Holton, James M]

In coot, select.

Validate > Difference Map Peaks > Find Peaks Above 4 r.m.s.d > [Find Peaks]

What do you see?

Always remember, the real-space representation of your Rwork is the Fo-Fc map.  
The biggest peak or valley in this map is usually dominating your Rwork/Rfree.  
If you feel your Rwork/Rfree are too high, it is best to think of something 
sensible to put into your biggest Fo-Fc peak.  If you have a big, negative 
feature in the middle of nowhere, try adjusting your bulk solvent parameters in 
refinement.

It is also a good idea to look at your data quality stats.  If I/sig(I) is 5, 
then I wouldn't expect Rwork/Rfree to ever drop below ~20%.  This is because 
the error in the data is already about 20%.  If your model fits better than 
that, it is probably modelling noise.

If you have no difference features to speak of and your Rwork/Rfree are still 
high, it can be a good thing to prune back your model to include only the atoms 
you are really sure about.  That is, well-ordered main chain, and maybe a few 
strong side chains like disulfides and obvious aromatics.  Refine this heavily 
pruned-model to convergence.  Don't worry about the R factors being high, they 
are supposed to be high when large parts of the model are missing.  By 
"convergence" I mean until the atoms stop moving.  R factors leveling off is 
not "convergence".  If your atoms are still wandering about and changing B 
factors, then run the refinement one more time.  Wait for things to settle.  
This is kind of like running a column, you want to wait until everything has 
equilibrated before you inject something new.  This kind of equilibration is 
the only way to know that the rise or fall in R you see is due to the change 
you just made, as opposed to an after-effect of something you did a few dozen 
cycles ago.  Once that is done, look at the top feature in your Fo-Fc 
difference map.  This tallest peak is the least likely thing in your unit cell 
to be wrong.  Build in this feature.  Then re-refine to convergence again.  
This can take a while, but it is the best way to ensure that you avoid model 
bias of any kind.  Building just one feature at a time is the most conservative 
strategy.  If you're in more of a hurry, you might consider anything above 6 
sigmas to be "safe", but 5 is pushing it, and 4 is dangerous unless there is 
nothing else left in the map.

Happy Building!

-James Holton
MAD Scientist

On 3/23/2019 9:17 PM, StrBio wrote:
ALL.

I have data at 2.4 A in P21 sp gr, helical protein.
Refined to Rwork 29 Rfree 34 with nice density map and all nice statistics 
oither Rfactor (by Phenix). Refmac quit same.
Should I deposit it or look better data?
Any suggestion?





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Re: [ccp4bb] Refinement

[Eleanor Dodson]

In Refmac there is a plot of Rfactors v resolution, Could you high
resolution data (or low for that matter) be particularly high?
Eleanor

On Sun, 24 Mar 2019 at 05:06, Lorenzo Briganti 
wrote:

> If you’re sure about your refinement parameters and results, I would check
> PDBredo just in case. I’d go for better Rs, but maybe it’s not possible
> with your data.
>
> Best,
>
> Lorenzo
>
>
> domingo, 24 de março de 2019 01:17 -0300 de biophysics.w...@gmail.com <
> biophysics.w...@gmail.com>:
>
> ALL.
>
> I have data at 2.4 A in P21 sp gr, helical protein.
> Refined to Rwork 29 Rfree 34 with nice density map and all nice statistics
> oither Rfactor (by Phenix). Refmac quit same.
> Should I deposit it or look better data?
> Any suggestion?
>
>
>
> --
>
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>
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>
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Re: [ccp4bb] Refinement

[Lorenzo Briganti]

If you’re sure about your refinement parameters and results, I would check 
PDBredo just in case. I’d go for better Rs, but maybe it’s not possible with 
your data.

Best,

Lorenzo


domingo, 24 de março de 2019 01:17 -0300 de biophysics.w...@gmail.com 
:
ALL.

I have data at 2.4 A in P21 sp gr, helical protein.
Refined to Rwork 29 Rfree 34 with nice density map and all nice statistics 
oither Rfactor (by Phenix). Refmac quit same.
Should I deposit it or look better data?
Any suggestion?





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Re: [ccp4bb] Refinement with native and anomalous data

[Piotr Wilk]

 >My advice is to simply cycle back-and-forth between the two data sets.
Refine against your high-resolution data until that converges, then simply
>switch to the other mtz file and use F+ F-, along with "anomalous
wavelength" and probably "refine orefine no", unless you want to refine the
>occupancies of Mn and other heavies.  Personally, I prefer to define
occupancy refinement explicitly using the REFMAC "occupancy group" etc.
>commands.  Once the SAD refinement has converged (as in positions,
occupancies, and B factors stop moving), then switch back to the high-   >
res.  Once that converges, shift back to anomalous again.  Then high-res,
then anomalous, then high-res, etc.

I haven't try that yet, but this definitely looks worth trying. I believe,
that this will give me a chance to use both sources of information
"sequentially" rather than "simultaneously". And what about rebuilding of
the model in-between refinement cycles (coot)?

>I understand that it is tempting to desire software that can somehow
incorporate data from disparate crystals and give you a single "right"
model, >but that can only happen if your data sets are highly isomorphous.
Think about it.  What if one data set has a serine side chain in the +
rotamer >and another data set has the same residue is - or trans ?  What
would you expect to find in your "right" model?

It is indeed tempting, but probably unrealistic as of today. Nevertheless
there is more and more parameters included in refinement protocols (for
example NMR data derived restraints, see
https://doi.org/10.1107/S2059798318000979). I admit that we only work with
the models which are only partly correct, but this should not stop us from
trying to get them ever better and newer protocols often aid this pursuit
(f.e. PDB_REDO).
In case of using data from two different crystals I'd expect the putative
serine to be in two alternative conformations with their occupancies
proportional to the strength of the signal from each crystal.
In my case the crystal belongs to C222(1) with 103.56 106.69 216.70 90.0
90.0 90.0 cell parameters, diffracting to ~1.85A. The crystal itself was a
big one with ~100um x 70um x 70um (on average). First the native data were
collected at 13.5keV with 2000 images x 0.1deg starting from the optimized
angle and resulted in redundancy of ~7.3 . Edge scan was performed and
beamline re-tuned to the peak of Mn edge. Second data set was 3600 x 0.1
deg from a different position of the same crystal (lucky to grow big ones).
The unit cell parameters differed by a fraction of an angstrom (as far as I
recall, but I will check exactly) so they are quite isomorphous. I believe
even more than some of the room-temperature data collected from multiple
crystals which were successful in phasing.

>Oscillating between data sets, however, allows you to have a well-defined
final refinement target, but for the things that are not well-defined by
>your final data set (such as anomalous signal or occupancy), you can "fill
in" those blank spaces by creating bias in the starting structure.

Isn't model bias what we usually try to minimize?

>In the present case, however, both data sets are from the same crystal, so
structural differences like rotamer shifts are not expected.  In fact, >why
not just merge the two data sets?  If you take your two sets of FP, put
them together with CAD, and scale them with SCALEIT, what is the >R-factor
between them?  If the R is less than 5-7% then there is seldom any reason
not to merge them, but if your two wavelengths are 10-15% >different you
should really ask yourself why.  Always a good idea to make an Fo-Fo map
and see where the largest difference peaks are.  If they >are on top of
heavy atoms then you might just be seeing the f' difference, and that is
OK, but you could also be seeing radiation-induced >changes, and those are
perhaps not what you are looking for?  Which data set was collected first?

Instead of merging I just copied some columns (these corresponding somehow
to anomalous differences) from the low energy mtz to the high energy one. I
can use this file sequentially alternating the refmac protocol, taking only
the relevant columns, but keeping the same Rfree flags.
I will also try merging both data sets for comparison. Admittedly I hope
for minimal radiation damage and there were no obvious indication for it
from the data processing, but I could check for it f.e. calculating Fo-Fo
map with first whatever 120deg- last 120deg. But this is a separate issue.

I will try both merging of the data sets and "alternating" protocol first
thing on Monday (I need my linux machine for it).

Thank you for advice,
Best regards,
  Piotrek

pt., 4 sty 2019 o 19:42 James Holton 
napisał(a):

> My advice is to simply cycle back-and-forth between the two data sets.
> Refine against your high-resolution data until that converges, then simply
> switch to the other mtz file and use F+ F-, along with "anomalous
> wavelength" and 

Re: [ccp4bb] Refinement with native and anomalous data

[James Holton]

My advice is to simply cycle back-and-forth between the two data sets.  
Refine against your high-resolution data until that converges, then 
simply switch to the other mtz file and use F+ F-, along with "anomalous 
wavelength" and probably "refine orefine no", unless you want to refine 
the occupancies of Mn and other heavies. Personally, I prefer to define 
occupancy refinement explicitly using the REFMAC "occupancy group" etc. 
commands.  Once the SAD refinement has converged (as in positions, 
occupancies, and B factors stop moving), then switch back to the 
high-res.  Once that converges, shift back to anomalous again.  Then 
high-res, then anomalous, then high-res, etc.


Obviously, you want to have the same Free-R flags in both mtz files.

You might think that all this back-and-forth would be redundant. Doesn't 
"refinement" of a given model and given data set always converge to the 
same final coordinate set?  No, it does not.  All you need to do is 
"jiggle" your model and re-refine and you will get something new.  RMSDs 
usually around 0.4 A or so, depending on resolution.  What this means 
pragmatically is that any given refinement has a lot of "slop" in its 
final result, and is therefore quite sensitive to initial conditions.  
You can therefore easily incorporate information from multiple data sets 
by doing this switching back-and-forth.  The information from one data 
set then manifests as "bias" for the start of the next refinement.  No 
special software required!


I understand that it is tempting to desire software that can somehow 
incorporate data from disparate crystals and give you a single "right" 
model, but that can only happen if your data sets are highly 
isomorphous.  Think about it.  What if one data set has a serine side 
chain in the + rotamer and another data set has the same residue is - or 
trans ?  What would you expect to find in your "right" model?


Oscillating between data sets, however, allows you to have a 
well-defined final refinement target, but for the things that are not 
well-defined by your final data set (such as anomalous signal or 
occupancy), you can "fill in" those blank spaces by creating bias in the 
starting structure.


In the present case, however, both data sets are from the same crystal, 
so structural differences like rotamer shifts are not expected.  In 
fact, why not just merge the two data sets?  If you take your two sets 
of FP, put them together with CAD, and scale them with SCALEIT, what is 
the R-factor between them?  If the R is less than 5-7% then there is 
seldom any reason not to merge them, but if your two wavelengths are 
10-15% different you should really ask yourself why.  Always a good idea 
to make an Fo-Fo map and see where the largest difference peaks are.  If 
they are on top of heavy atoms then you might just be seeing the f' 
difference, and that is OK, but you could also be seeing 
radiation-induced changes, and those are perhaps not what you are 
looking for?  Which data set was collected first?


-James Holton
MAD Scientist


On 1/4/2019 2:16 AM, Steiner, Roberto wrote:

Dear Piotrek

You can find some info on the use of prior phase information in 
refinement with Refmac in


Acta Crystallogr D Biol Crystallogr. 
 2011 
Apr;67(Pt 4):355-67. doi: 10.1107/S0907444911001314. Epub 2011 Mar 18.



  REFMAC5 for the refinement of macromolecular crystal structures.

Murshudov GN 
1, 
Skubák P 
, 
Lebedev AA 
, 
Pannu NS 
, 
Steiner RA 
, 
Nicholls RA 
, 
Winn MD 
, 
Long F 
, 
Vagin AA 
.


and references therein.
As stated in the paper (paragraph 2.2.2), the incorporation of prior 
phase information by the refinement function is especially useful in 
the early and middle stages of model building and at all stages of 
structure solution at lower resolutions, owing to the improvement in 
the observation-to-parameter ratio.
Refinement in Refmac is very fast therefore the best thing (as you 
just did) is to try both options and see.


With best wishes
Roberto



On 4 Jan 2019, at 09:55, Piotr Wilk > wrote:


Dear Eleanor,

I have used the ACORN previously for structure solution but I will 

Re: [ccp4bb] Refinement with native and anomalous data

[Steiner, Roberto]

Dear Piotrek

You can find some info on the use of prior phase information in refinement with 
Refmac in

Acta Crystallogr D Biol 
Crystallogr. 2011 
Apr;67(Pt 4):355-67. doi: 10.1107/S0907444911001314. Epub 2011 Mar 18.
REFMAC5 for the refinement of macromolecular crystal structures.
Murshudov 
GN1,
 Skubák 
P,
 Lebedev 
AA,
 Pannu 
NS,
 Steiner 
RA,
 Nicholls 
RA,
 Winn 
MD,
 Long 
F,
 Vagin 
AA.

and references therein.

As stated in the paper (paragraph 2.2.2), the incorporation of prior phase 
information by the refinement function is especially useful in the early and 
middle stages of model building and at all stages of structure solution at 
lower resolutions, owing to the improvement in the observation-to-parameter 
ratio.
Refinement in Refmac is very fast therefore the best thing (as you just did) is 
to try both options and see.

With best wishes
Roberto



On 4 Jan 2019, at 09:55, Piotr Wilk 
mailto:wilk.piot...@gmail.com>> wrote:

Dear Eleanor,

I have used the ACORN previously for structure solution but I will have to read 
more about its functionality in structure refinement.
I have run two Refmac jobs using either native or anomalous data with otherwise 
default parameters resulting in R/Rfree of 0.2013/0.2458 and 0.1862/0.2274 
respectively for crystal diffracting to ~1.85A. This seems to me, that using 
anomalous signal in refinement can be useful at least in some cases.

Regards,
  Piotrek


Hmm - you can certainly generate "MAD" phases using the anom signal from one 
data set, and then PARROT or some such density modification tool to extend 
those phases for the higher resolution reflections..
Or ACORN can work well if he data resolution is high enough to give good phases 
for all the data


Then you can use those phases in the initial refinement procedure - the usual 
idea is to use them till the R factor drops below 30% or 35% then just refine 
against the Fobs, phasing just from the model .

But I dont think it is ever worth working at a limited resolution..


Dear Eleanor,

thank you for your comment. My crystals of interest diffract with dmin usually 
between 1.4 and 1.9 A (in high energy data set) and significant anomalous 
signal extends usually to approx. 4 A (in low energy data set).
Certainly I do agree, that in "EITHER, OR" situation one can check both 
approaches, compare the results and take the more convincing one. I can easily 
do that in Refmac running one job against data with Friedel pairs merged and 
parallel one against data with Friedel pairs unmerged. I was considering rather 
an "AND" scenario in which in addition to high resolution data (FP) I'd include 
information from anomalous signal (F+ F-, DANO). I understand that this should 
increase number of observations from a given sample and therefore help to 
refine positions, occupancies and perhaps ADPs for at least a fraction of atoms 
in a model (Mn ions and S in my case). I imagine it as somehow analogical to 
adding geometrical restrains derived from very high resolution data to 
refinement protocols.
I was wondering first of all if my reasoning is sensible and if there is an 
existing protocol to try this?

With kind regards,
 Piotrek

czw., 3 sty 2019 o 22:00 Eleanor Dodson 
mailto:eleanor.dod...@york.ac.uk>> napisał(a):
Hmm - you can certainly generate "MAD" phases using the anom signal from one 
data set, and then PARROT or some such density modification tool to extend 
those phases for the higher resolution reflections..
Or ACORN can work well if he data resolution is high enough to give good phases 
for all the data


Then you can use those phases in the initial refinement procedure - the usual 
idea is to use them till the R factor drops below 30% or 35% then just refine 
against the Fobs, phasing just from the model .

But I dont think it is ever worth working at a limited resolution..

eleanor

On Thu, 3 Jan 2019 at 20:40, Piotr Wilk 
mailto:wilk.piot...@gmail.com>> wrote:
Dear Eleanor,

thank you for your comment. My crystals of interest diffract with dmin usually 
between 1.4 and 1.9 A (in high energy data set) and significant anomalous 
signal 

Re: [ccp4bb] Refinement with native and anomalous data

[Piotr Wilk]

Dear Eleanor,

I have used the ACORN previously for structure solution but I will have to
read more about its functionality in structure refinement.
I have run two Refmac jobs using either native or anomalous data with
otherwise default parameters resulting in R/Rfree of 0.2013/0.2458 and
0.1862/0.2274 respectively for crystal diffracting to ~1.85A. This seems to
me, that using anomalous signal in refinement can be useful at least in
some cases.

Regards,
  Piotrek


Hmm - you can certainly generate "MAD" phases using the anom signal from
one data set, and then PARROT or some such density modification tool to
extend those phases for the higher resolution reflections..
Or ACORN can work well if he data resolution is high enough to give good
phases for all the data


Then you can use those phases in the initial refinement procedure - the
usual idea is to use them till the R factor drops below 30% or 35% then
just refine against the Fobs, phasing just from the model .

But I dont think it is ever worth working at a limited resolution..


Dear Eleanor,

thank you for your comment. My crystals of interest diffract with dmin
usually between 1.4 and 1.9 A (in high energy data set) and significant
anomalous signal extends usually to approx. 4 A (in low energy data set).
Certainly I do agree, that in "EITHER, OR" situation one can check both
approaches, compare the results and take the more convincing one. I can
easily do that in Refmac running one job against data with Friedel pairs
merged and parallel one against data with Friedel pairs unmerged. I was
considering rather an "AND" scenario in which in addition to high
resolution data (FP) I'd include information from anomalous signal (F+ F-,
DANO). I understand that this should increase number of observations from a
given sample and therefore help to refine positions, occupancies and
perhaps ADPs for at least a fraction of atoms in a model (Mn ions and S in
my case). I imagine it as somehow analogical to adding geometrical
restrains derived from very high resolution data to refinement protocols.
I was wondering first of all if my reasoning is sensible and if there is an
existing protocol to try this?

With kind regards,
 Piotrek

czw., 3 sty 2019 o 22:00 Eleanor Dodson 
napisał(a):

> Hmm - you can certainly generate "MAD" phases using the anom signal from
> one data set, and then PARROT or some such density modification tool to
> extend those phases for the higher resolution reflections..
> Or ACORN can work well if he data resolution is high enough to give good
> phases for all the data
>
>
> Then you can use those phases in the initial refinement procedure - the
> usual idea is to use them till the R factor drops below 30% or 35% then
> just refine against the Fobs, phasing just from the model .
>
> But I dont think it is ever worth working at a limited resolution..
>
> eleanor
>
> On Thu, 3 Jan 2019 at 20:40, Piotr Wilk  wrote:
>
>> Dear Eleanor,
>>
>> thank you for your comment. My crystals of interest diffract with dmin
>> usually between 1.4 and 1.9 A (in high energy data set) and significant
>> anomalous signal extends usually to approx. 4 A (in low energy data set).
>> Certainly I do agree, that in "EITHER, OR" situation one can check both
>> approaches, compare the results and take the more convincing one. I can
>> easily do that in Refmac running one job against data with Friedel pairs
>> merged and parallel one against data with Friedel pairs unmerged. I was
>> considering rather an "AND" scenario in which in addition to high
>> resolution data (FP) I'd include information from anomalous signal (F+ F-,
>> DANO). I understand that this should increase number of observations from a
>> given sample and therefore help to refine positions, occupancies and
>> perhaps ADPs for at least a fraction of atoms in a model (Mn ions and S in
>> my case). I imagine it as somehow analogical to adding geometrical
>> restrains derived from very high resolution data to refinement protocols.
>> I was wondering first of all if my reasoning is sensible and if there is
>> an existing protocol to try this?
>>
>> With kind regards,
>>  Piotrek
>>
>> czw., 3 sty 2019 o 17:02 Eleanor Dodson 
>> napisał(a):
>>
>>> I think any decision depends on the resolution of your two data sets. If
>>> they are very different I would choose the higher resolution one.
>>>
>>> If that is the Anom data then I would use the anom signal at least in
>>> the first cycles to improve the phases..
>>>
>>> Eleanor
>>>
>>> On Thu, 3 Jan 2019 at 14:59, Piotr Wilk  wrote:
>>>
 Dear CCP4 experts,

 I'd like to ask your opinion about using anomalous signal in refinement
 of crystal structures in addition to using high resolution native data.
 I am working on a series of structures for which I have collected two
 data sets (from the same crystal):
 1 - native with higher resolution
 2 - anomalous at MN 

Re: [ccp4bb] Refinement with native and anomalous data

[Eleanor Dodson]

I think any decision depends on the resolution of your two data sets. If
they are very different I would choose the higher resolution one.

If that is the Anom data then I would use the anom signal at least in the
first cycles to improve the phases..

Eleanor

On Thu, 3 Jan 2019 at 14:59, Piotr Wilk  wrote:

> Dear CCP4 experts,
>
> I'd like to ask your opinion about using anomalous signal in refinement of
> crystal structures in addition to using high resolution native data.
> I am working on a series of structures for which I have collected two data
> sets (from the same crystal):
> 1 - native with higher resolution
> 2 - anomalous at MN absorption edge peak.
> The structures were solved with MR and preliminary refinement using the
> native data only yields decent statistics, but I also use anomalous data to
> verify presence and position of manganese ions. For this I used ANODE which
> lists four strong peaks (~30 sigma) as expected for manganese ions and
> around 45 weaker peaks (~9-5 sigma) for sulfur atoms in Cys and Met. I am
> happy to use this information in model building but I was also wondering if
> (and how) beneficial would it be to use both high resolution structure
> factors and somehow lower resolution yet highly specific anomalous signal
> in the same round of refinement?
> In Refmac5 I can use either refinement with "no prior phase information"
> taking FP and SIGFP or "SAD data directly" with SIGFP F(+) SIGF(+) F(-)
> SIGF(-), but I didn't find any "MAD" option to use both.
> I have the following columns in my mtz files:
> for native data: H K L FP SIGFP FreeRflag
> for anomalous data : H K L FP SIGFP F(+) SIGF(+) F(-) SIGF(-) FreeRflag
> or :   H K L FP SIGFP DANO SIGDANO ISYM FreeRflag
>
> I could use CAD to merge the interesting columns into a single mtz file
> containing:
>  H K L FP SIGFP FreeRflag F(+) SIGF(+) F(-) SIGF(-) DANO SIGDANO
>
> I'd appreciate any comments or advise how to use both sources of
> information in the refinement.
>
> I wish you all a Happy New Year.
> Kind regards,
> Piotrek
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] refinement papers

[Oleg Sobolev]

Hi Careina,

On behalf of Pavel Afonine:

This (and references inside) may be a start:

http://scripts.iucr.org/cgi-bin/paper?S0907444912001308

Then may be you could reuse some of existing presentations from here

https://www.phenix-online.org/presentations/

Best regards,
Oleg Sobolev.

On Mon, Jun 25, 2018 at 5:21 AM, Careina Edgooms <
02531c126adf-dmarc-requ...@jiscmail.ac.uk> wrote:

> Hello everyone
>
> I am giving talk about refinement and model building. I wonder if anyone
> knows of latest relevant papers on this topic that you could suggest for me?
> thank you
>
> Careina
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] refinement papers

[Misba Ahmad]

Here are some nice papers:

journals.iucr.org/d/issues/2012/04/00/


Best
Misbha


On Mon, 25 Jun 2018 at 14:26, Careina Edgooms <
02531c126adf-dmarc-requ...@jiscmail.ac.uk> wrote:

> Hello everyone
>
> I am giving talk about refinement and model building. I wonder if anyone
> knows of latest relevant papers on this topic that you could suggest for me?
> thank you
>
> Careina
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] Refinement at 4A resolution

[Christian Roth]

Hi Appu,

you start already with a fixed spacegroup (scaled merged data) according 
to your pointless log. So you can't get another possible solution from 
pointless.


Cheers


Am 22.04.2015 um 22:28 schrieb Appu kumar:

Dear CCP4 Member,
I seek your advice on the refinement issues at the low resolution 4A. I
am trying to refine a membrane protein structure after getting the
phases from MR using the PHASER. The soluble domain structure which
comprises of 40% of protein has been used as template (sequence identity
80%) in MR search . The PHASER gave  a good solution having TFZ value of
about 14.3. I have then created the polyA model for the transmembrane
domain from distant homolog which share 30% sequence identity for TM
region and try to find the phases for whole TM domain keeping the
soluble domain fixed. I got lucky in getting the phases for the whole
protein using the PHASER (TFZ=17.6) but the during the refinement, Rwork
and Rfree got stalled at the 41 and 44 respectively after several cycle
of the refinement in both refmac and phenix. I checked the spacegroup
with pointless and it suggests C2221. I have attached the pointless and
phenix.xtriage run file with this mail for your evaluation.
Phenix.xtriage suggests no major pathologies with the data except the
mild psuedomerohedral twining. There are two molecules of protein in
ASU. Evaluation of the density maps, suggest reasonable map for the most
of protein part. I am wondering why Rwork and Rfree are not coming down
despite of the good MR solution and what i am doing wrong with
refinement and if there is some pathologies associated with the data
which needs to be answered before heading to refinement.

Thanks for your help in advance.

Appu

Re: [ccp4bb] Refinement at 4A resolution

[Phil Evans]

The Xtriage  Pointless logs don't show definitively that the space group is 
C2221 as they have been run on the merged data. You may need to check them with 
the unmerged data, and perhaps run molecular replacement in a lower symmetry 
such as C2

Phil

On 22 Apr 2015, at 22:28, Appu kumar appu.kum...@gmail.com wrote:

 Dear CCP4 Member,
 I seek your advice on the refinement issues at the low resolution 4A. I am 
 trying to refine a membrane protein structure after getting the phases from 
 MR using the PHASER. The soluble domain structure which comprises of 40% of 
 protein has been used as template (sequence identity 80%) in MR search . The 
 PHASER gave  a good solution having TFZ value of about 14.3. I have then 
 created the polyA model for the transmembrane domain from distant homolog 
 which share 30% sequence identity for TM region and try to find the phases 
 for whole TM domain keeping the soluble domain fixed. I got lucky in getting 
 the phases for the whole protein using the PHASER (TFZ=17.6) but the during 
 the refinement, Rwork and Rfree got stalled at the 41 and 44 respectively 
 after several cycle of the refinement in both refmac and phenix. I checked 
 the spacegroup with pointless and it suggests C2221. I have attached the 
 pointless and phenix.xtriage run file with this mail for your evaluation. 
 Phenix.xtriage suggests no major pathologies with the data except the mild 
 psuedomerohedral twining. There are two molecules of protein in ASU. 
 Evaluation of the density maps, suggest reasonable map for the most of 
 protein part. I am wondering why Rwork and Rfree are not coming down despite 
 of the good MR solution and what i am doing wrong with refinement and if 
 there is some pathologies associated with the data which needs to be answered 
 before heading to refinement.  
 
 Thanks for your help in advance. 
 
 Appu
 
 93_pointless.logxtriage_50.log

Re: [ccp4bb] Refinement at 4A resolution

[Appu kumar]

Dear All,
Sorry for the wrong pointless file. With this mail i have attached the
pointless run file from the unmerged data.  This file also suggests the
C2221 spacegroup.
Appu

On 22 April 2015 at 17:40, Christian Roth christian.r...@bbz.uni-leipzig.de
 wrote:

 Hi Appu,

 you start already with a fixed spacegroup (scaled merged data) according
 to your pointless log. So you can't get another possible solution from
 pointless.

 Cheers



 Am 22.04.2015 um 22:28 schrieb Appu kumar:

 Dear CCP4 Member,
 I seek your advice on the refinement issues at the low resolution 4A. I
 am trying to refine a membrane protein structure after getting the
 phases from MR using the PHASER. The soluble domain structure which
 comprises of 40% of protein has been used as template (sequence identity
 80%) in MR search . The PHASER gave  a good solution having TFZ value of
 about 14.3. I have then created the polyA model for the transmembrane
 domain from distant homolog which share 30% sequence identity for TM
 region and try to find the phases for whole TM domain keeping the
 soluble domain fixed. I got lucky in getting the phases for the whole
 protein using the PHASER (TFZ=17.6) but the during the refinement, Rwork
 and Rfree got stalled at the 41 and 44 respectively after several cycle
 of the refinement in both refmac and phenix. I checked the spacegroup
 with pointless and it suggests C2221. I have attached the pointless and
 phenix.xtriage run file with this mail for your evaluation.
 Phenix.xtriage suggests no major pathologies with the data except the
 mild psuedomerohedral twining. There are two molecules of protein in
 ASU. Evaluation of the density maps, suggest reasonable map for the most
 of protein part. I am wondering why Rwork and Rfree are not coming down
 despite of the good MR solution and what i am doing wrong with
 refinement and if there is some pathologies associated with the data
 which needs to be answered before heading to refinement.

 Thanks for your help in advance.

 Appu


#CCP4I VERSION CCP4Interface 2.2.1
#CCP4I SCRIPT LOG pointless
#CCP4I DATE 22 Apr 2015  17:39:30
#CCP4I USER appu
#CCP4I PROJECT AS015crv6ctd2nq
#CCP4I JOB_ID 119
#CCP4I SCRATCH /tmp/appu
#CCP4I HOSTNAME sasha
#CCP4I PID 47982

 
 ###
 ###
 ###
 ### CCP4 6.4: POINTLESS version 1.9.16 : 21/08/14##
 ###
 User: appu  Run date: 22/ 4/2015 Run time: 17:39:30 


 Please reference: Collaborative Computational Project, Number 4. 1994.
 The CCP4 Suite: Programs for Protein Crystallography. Acta Cryst. D50, 760-763.
 as well as any specific reference in the program write-up.

 Input command lines 

HKLIN /home/appu/xtal/2015_04_17_APS_24IDC/AS015c/imosflm/AS115c_1_0001.mtz
HKLOUT /home/appu/xtal/2015_04_17_APS_24IDC/AS015c/refine/AS115c_1_0001_pointless1.mtz
## This script run with the command   ##
# /home/appu/Downloads/ccp4-6.4.0/bin/pointless


 End of input

OS type:  linux
Release Date: 21st August2014


**
**
* POINTLESS  *
*   1.9.16   *
**
*   Determine Laue group from unmerged intensities   *
* Phil Evans MRC LMB, Cambridge  *
* Uses cctbx routines by Ralf Grosse-Kunstleve et al.*
**
**


 Spacegroup information obtained from library file: 
 Logical Name: SYMINFO   Filename: /home/appu/Downloads/ccp4-6.4.0/lib/data/syminfo.lib


Reflection list generated from file:
/home/appu/xtal/2015_04_17_APS_24IDC/AS015c/imosflm/AS115c_1_0001.mtz

Title: Untitled

   Space group from HKLIN file : C 2 2 21
   Cell:  130.42 213.36 160.80  90.00  90.00  90.00
   Resolution range in file: 65.213.75

Time for reading file(s):2.190 secs

===

* Summary of test data read in:
   Resolution range accepted:65.213.75

   Number of reflections  = 23371
   Number of observations =150865
   Number of parts=619082
   Number of batches in file  =   600
   Number of datasets = 1
  Project: New Crystal: New Dataset: New
  Run number:   1 consists of batches  1 to600
 Resolution range for run:65.213.75
 Phi range: 0.00 to   180.00   Time range: 0.00 to   180.00
 Closest reciprocal axis to 

Re: [ccp4bb] Refinement with phase/FOM and HL coefficients

[Eleanor Dodson]

Potentially there is more information in the 4 parameters HLA HLB HLC HLD
which can describe a bi-modal distribution of phase probabilities( that
would be generated by experimental phasing)  than can be carried by the 2
parameter PHI FOM which will describe a uni-modal distribution. However if
HLC=HLD=0.0 as will be so if the phase estimates are derived from a
structure factor calculation the two options are equivalent .



On 17 February 2015 at 09:14, Mohamed Noor mohamed.n...@staffmail.ul.ie
wrote:

 Dear all

 In a few places (Refmac and Phenix, maybe there are also others), there is
 an option to use either phase/FOM or HL for refinement and DM. Is there any
 difference between these two? The dataset in question is a Fe SAD dataset.

 Thanks.
 Mohamed

Re: [ccp4bb] Refinement with refmac05 and methionine density

[Greg Costakes]

Hi Jeorge,

The simplest answer is that you have multiple positions for the Methionine. So 
you can try adding in an alternate position for it. However, you didn’t mention 
the sigma cutoff or e-/A^3 for either of your density maps. Its quite possible 
that your fofc map is just scaled way down and what you are seeing is an 
exaggeration of what’s barely there. Hope this helps. Cheers!

- Greg

---
Greg Costakes, Ph.D.
Department of Biochemistry
University of Cambridge
80 Tennis Court Road
Cambridge, CB2 1GA
United Kingdom



From: jeorgemarley thomas
Sent: Wednesday, November 12, 2014 8:16 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Refinement with refmac05 and methionine density

Dear All,

I am refining the structure with refmac05, and after each refinement the 
density around this methionine is flipping. and it is difficult to say where 
this methionine actually have the density. please suggest what I should I do. I 
am attaching the snap here.

Thanks in Advance for your kind suggestions

Jeorge



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Re: [ccp4bb] Refinement with refmac05 and methionine density

[Eleanor Dodson]

That looks really odd - the whole MET has a negative/positive ghost


Second conformations usually branch at the CB, and dont look like that.

Are you sure you havent somehow got two MET residues in the coordinate set
? Turn on cysmmetry display in coot and look for clashes around there.

Does the rest of the map look OK?

I would set the occupancies of the MET and nearby side chains to 0.00 (you
can do that in coot) then refine and look at the maps again.

This is very old fashioned but you can do this:
distang xyzin coords.pdb
dist inter
end

to get a list of close symmetry clashes  contacts
Eleanor


On 12 November 2014 09:17, Greg Costakes gcost...@purdue.edu wrote:

   Hi Jeorge,

 The simplest answer is that you have multiple positions for the
 Methionine. So you can try adding in an alternate position for it. However,
 you didn’t mention the sigma cutoff or e-/A^3 for either of your density
 maps. Its quite possible that your fofc map is just scaled way down and
 what you are seeing is an exaggeration of what’s barely there. Hope this
 helps. Cheers!

 - Greg


 ---
 Greg Costakes, Ph.D.
 Department of Biochemistry
 University of Cambridge
 80 Tennis Court Road
 Cambridge, CB2 1GA
 United Kingdom

 

  *From:* jeorgemarley thomas kirtswab...@gmail.com
 *Sent:* Wednesday, November 12, 2014 8:16 AM
 *To:* CCP4BB@JISCMAIL.AC.UK
 *Subject:* [ccp4bb] Refinement with refmac05 and methionine density

  Dear All,

 I am refining the structure with refmac05, and after each refinement the
 density around this methionine is flipping. and it is difficult to say
 where this methionine actually have the density. please suggest what I
 should I do. I am attaching the snap here.

 Thanks in Advance for your kind suggestions

 Jeorge




 --
http://www.avast.com/

 This email is free from viruses and malware because avast! Antivirus
 http://www.avast.com/ protection is active.

Re: [ccp4bb] Refinement with refmac05 and methionine density

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Jeorge,

the 'ghost' could be explained by a shift of an entire section, i.e.
you have to split several residues into A+B, not only the MET.

The cylR2 structure in 2XJ3 is one example where this was necessary.

Best,
Tim

On 11/12/2014 11:23 AM, Eleanor Dodson wrote:
 That looks really odd - the whole MET has a negative/positive
 ghost
 
 
 Second conformations usually branch at the CB, and dont look like
 that.
 
 Are you sure you havent somehow got two MET residues in the
 coordinate set ? Turn on cysmmetry display in coot and look for
 clashes around there.
 
 Does the rest of the map look OK?
 
 I would set the occupancies of the MET and nearby side chains to
 0.00 (you can do that in coot) then refine and look at the maps
 again.
 
 This is very old fashioned but you can do this: distang xyzin
 coords.pdb dist inter end
 
 to get a list of close symmetry clashes  contacts Eleanor
 
 
 On 12 November 2014 09:17, Greg Costakes gcost...@purdue.edu
 wrote:
 
 Hi Jeorge,
 
 The simplest answer is that you have multiple positions for the 
 Methionine. So you can try adding in an alternate position for
 it. However, you didn’t mention the sigma cutoff or e-/A^3 for
 either of your density maps. Its quite possible that your fofc
 map is just scaled way down and what you are seeing is an
 exaggeration of what’s barely there. Hope this helps. Cheers!
 
 - Greg
 
 
 ---

 
Greg Costakes, Ph.D.
 Department of Biochemistry University of Cambridge 80 Tennis
 Court Road Cambridge, CB2 1GA United Kingdom
 
 


 
*From:* jeorgemarley thomas kirtswab...@gmail.com
 *Sent:* Wednesday, November 12, 2014 8:16 AM *To:*
 CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] Refinement with
 refmac05 and methionine density
 
 Dear All,
 
 I am refining the structure with refmac05, and after each
 refinement the density around this methionine is flipping. and it
 is difficult to say where this methionine actually have the
 density. please suggest what I should I do. I am attaching the
 snap here.
 
 Thanks in Advance for your kind suggestions
 
 Jeorge
 
 
 
 
 -- http://www.avast.com/
 
 This email is free from viruses and malware because avast!
 Antivirus http://www.avast.com/ protection is active.
 
 
 

- -- 
- --
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A

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iD8DBQFUY0O/UxlJ7aRr7hoRArkIAJsHP6gLpSkRtJPEE7/P/R453uGvJACeMfus
jFS3EEDy7g7ss5A6L3D3cco=
=uLjH
-END PGP SIGNATURE-

Re: [ccp4bb] Refinement of Iron-sulphur clusters

[Oliver Smart]

on 13/10/14 6:33 PM, Pedro Matias mat...@itqb.unl.pt wrote:

 In relation to this topic, I'd like to mention that SF4 has replaced FS4
 as the Fe4S4 monomer in the CCP4 monomer library.

 However, the dictionary values for bond lengths and angles are not
 correct, and this is especially noticeable when the dictionary is used
 in a high-resolution refinement (e.g., 1.05 A). In particular, the
 Fe-S-Fe angles are all set to 90 degrees, when in fact some are smaller
 and others are larger.

 This introduces a bias in the final rms angle deviation values. Fe4S4 is
 a distorted cubane, not a perfect cube.


Pedro,

Yes you are 100% correct

The dictionaries I sent have values that are obtained from an analysis of
small
molecule crystal structures in the CSD (by hand with conquest). For
instance
the Fe-Scluster-Fe angle has a mean of  73.4 degree with a standard
deviation
of 0.7 degrees. The restraints are compatible with Engh and Huber restraints
you
will be using for your protein and for that matter the Parkinson et al
restraints used
for any nucleic acid (because they come from a similar data mining
procedure).
So if Stephen used this dictionary he would not  introduce a bias in the
final rms
angle deviation value because the restraint values would be sensible.

If you to check the restraint values are sensible then you check them
against atomic
resolution structures for instance
http://www.rcsb.org/pdb/explore/explore.do?structureId=1B0Y
at 0.93 Å resolution structure determined by George Sheldrick. This has SF4
with
Fe-Scluster-Fe angles around 73.3 degrees.

I would strongly advise anyone against using a restraint dictionary for
Fe4S4 that uses
90 degree as the ideal angle because you are restraining towards values that

make no sense. If the dictionary contains chiral volume terms then I would
ask the
question why? But this is just my personal take.

If CCP4 would like to redistribute/work the dictionaries sent then we would
be happy
(please email off-list).

Dr Oliver Smart
Director SmartSci Limited http://www.smartsci.uk/
 Consultant Global Phasing Ltd http://www.globalphasing.com/

Re: [ccp4bb] Refinement of Iron-sulphur clusters

[George M. Sheldrick]

Dear Oliver,

Since you mentioned my name let me try to confuse the issue. The iron
atoms in Fe4S4 clusters can adopt different oxidation states, e.g. in
HiPIPs and Ferrodoxins, and one might expect this to influence the
geometry of the clusters. So maybe you would need several different sets
of restraints. However at the time most of these structures were
determined, the influence of radiation damage was generally
underestimated. In general the first thing that happens in a synchrotron
beam is that the clusters mop up the electrons released by the action of
the radiation and so they probably had lower oxidation states than the
people determining the structures (at least this one) thought that they
had. What we should have done was to try to get a Moessbauer spectrum of
each crystal before and after the data collection, but it is easy to say
that now and anyway the crystals were probably too small.

Best wishes, George

 
 The dictionaries I sent have values that are obtained from an analysis of
 small
 molecule crystal structures in the CSD (by hand with conquest). For
 instance
 the Fe-Scluster-Fe angle has a mean of  73.4 degree with a standard
 deviation
 of 0.7 degrees. The restraints are compatible with Engh and Huber restraints
 you
 will be using for your protein and for that matter the Parkinson et al
 restraints used
 for any nucleic acid (because they come from a similar data mining
 procedure).
 So if Stephen used this dictionary he would not  introduce a bias in the
 final rms
 angle deviation value because the restraint values would be sensible.
 
 If you to check the restraint values are sensible then you check them
 against atomic
 resolution structures for instance
 http://www.rcsb.org/pdb/explore/explore.do?structureId=1B0Y
 at 0.93 Å resolution structure determined by George Sheldrick. This has SF4
 with
 Fe-Scluster-Fe angles around 73.3 degrees.
 
 I would strongly advise anyone against using a restraint dictionary for
 Fe4S4 that uses
 90 degree as the ideal angle because you are restraining towards values that
 
 make no sense. If the dictionary contains chiral volume terms then I would
 ask the
 question why? But this is just my personal take.
 
 If CCP4 would like to redistribute/work the dictionaries sent then we would
 be happy
 (please email off-list).
 
 Dr Oliver Smart
 Director SmartSci Limited http://www.smartsci.uk/
  Consultant Global Phasing Ltd http://www.globalphasing.com/
 

-- 
Prof. George M. Sheldrick FRS
Dept. Structural Chemistry,
University of Goettingen,
Tammannstr. 4,
D37077 Goettingen, Germany
Tel. +49-551-39-33021 or -33068
Fax. +49-551-39-22582

Re: [ccp4bb] Refinement of Iron-sulphur clusters

[Oliver Smart]

Stephen,

Robbie advice is 100% correct. Be careful about the naming of the sulphur
atoms (is S1 opposite S4 or next to it). I recall that the distributed CCP4
dictionaries have different atom naming than the PDB chemical components
dictionary.  If this is the problem is that the naming is different this
should result
in large restraint violations that should be appear in the REFMAC output
(others can tell you where).

In general diffraction from Fe S clusters is so strong that the atom
positions
are pretty much determined from the electron density. But in case it helps
I did some work data mining restraints for Fe2S2 and Fe4S4 from CSD
structures (this has to be done manually). Please find attached the
resulting FES.cif and SF4.cif restraint dictionaries (distributed with
BUSTER).

Good luck,

Oliver
---
Dr Oliver Smart
Director SmartSci Limited http://www.smartsci.uk/
 Consultant Global Phasing Ltd http://www.globalphasing.com/

on 10/10/14 5:33 PM, Robbie Joosten robbie_joos...@hotmail.com wrote:

 Dear Stephen,

 The dictionary is very specific about atom names. If you have them swapped
 (as many PDB entries do). The angle and chiral volume restraints will
wreck
 your cluster. You also need to make sure to provide LINK records to attach
 the cluster to the surrounding cysteines.

 HTH,
 Robbie

 Sent from my Windows Phone
 
 Van: Stephen Carr
 Verzonden: 10-10-2014 18:07
 Aan: CCP4BB@JISCMAIL.AC.UK
 Onderwerp: [ccp4bb] Refinement of Iron-sulphur clusters

 Dear CCP4 BBers,

 I am currently refining a model of a protein containing three iron sulphur
 clusters using Refmac (v5.8.0078) and am getting some unusual results.
One
 of the clusters (3fe 4s) seems to behave resonably and the refined
electron
 density looks very nice.  The atoms in the others clusters (4Fe4S and
 4Fe3S), however, migrate towards the centre of the cluster producing Fo-Fc
 difference maps with strong negative density at the centre surrounded by
 blobs of positive density where the atoms should be.

 I have checkedthe refmac dictionaries and there are entries for each of
the
 clusters, so I shouldn't have to explicitly include a cif file for the
 clusters as an input right?  In fact, when I did try to include a
dictionary
 file for one of the clusters refmac gave a warning message in the log
about
 duplicate monomers dictionaries and it made no difference to the
 refinement/maps anyway.

 On checking the refmac log file there is no specific mention that there
are
 hetero-groups in the pdb file and no explicit mention that it is using any
 dictionaries during the refinement.  How can I tell if refmac is
 reading/using the dictionaries?  Also bonds within the clusters are
flagged
 up as outliers and deviating by more than 10 sigma from ideal suggesting
 that they are not getting read?

 I realise that something chemically odd could be going on at the centres,
 but if I omit them from the model and calculate maps very strong density
 comes back around where the atoms sat before refinement, so I am pretty
 confident that they are in the right place to start with.

 Any help with this problem would be greatly appreciated, I'm sure there is
 something obvious that I am missing but can't see what that is at the
 moment.  It is particularly confusing since one cluster apparently behaves
 while the others do not.  I could, of course, try phenix or buster, but I
 would like to get to the bottom of the problem with refmac if possible.

 Thanks very much in advance,

 Steve

 Dr Stephen Carr
 Research Complex at Harwell (RCaH)
 Rutherford Appleton Laboratory
 Harwell Oxford
 Didcot
 Oxon OX11 0FA
 United Kingdom
 Email stephen.c...@rc-harwell.ac.uk
 tel 01235 567717
 This email and any attachments may contain confidential, copyright and or
 privileged material, and are for the use of the intended addressee only.
If
 you are not the intended addressee or an authorized recipient of the
 addressee, please notify us of receipt by returning the e-mail and do not
 use, copy, retain, distribute or disclose the information in or attached
to
 this email.

 Any views or opinions presented are solely those of the author and do not
 necessarily represent those of the Research Complex at Harwell.

 There is no guarantee that this email or any attachments are free from
 viruses and we cannot accept liability for any damage which you may
sustain
 as a result of software viruses which may be transmitted in or with the
 message.

 We use an electronic filing system. Please send electronic versions of
 documents, unless paper is specifically requested.

 This email may have a protective marking, for an explanation, please see:


http://www.mrc.ac.uk/About/informationandstandards/documentmarking/index.htm
 .





FES.cif
Description: CIF chemical test


SF4.cif
Description: CIF chemical test

Re: [ccp4bb] Refinement of Iron-sulphur clusters

[Stephen Carr]

Dear Matthew, Robbie Hans and Oliver,

Thanks for the advice, it seems that it is simply the geometry definitions in 
the dictionaries distributed with CCP4 6.4 are different to those calculated 
from the atomic coordinates.  For example, the chiral volume definitions in the 
for the 4Fe4S cluster are the opposite sign to those in my protein.  So 
swapping the atom names has sorted that, I am still working on the other 
clusters, but progress is being made.

Since I solved the structure by MR using coordinates downloaded from the pdb, I 
assumed the atom names would agree with the geometry definitions in Refmac 
(since the search model was also refined with refmac).  Have the cif 
dictionaries been modified/updated recently? 

thanks again,

Steve 

Dr Stephen Carr
Research Complex at Harwell (RCaH)
Rutherford Appleton Laboratory
Harwell Oxford
Didcot
Oxon OX11 0FA
United Kingdom
Email stephen.c...@rc-harwell.ac.uk
tel 01235 567717


From: Oliver Smart [osm...@smartsci.uk]
Sent: 13 October 2014 12:09
To: ccp4bb
Subject: Re: [ccp4bb] Refinement of Iron-sulphur clusters

Stephen,

Robbie advice is 100% correct. Be careful about the naming of the sulphur
atoms (is S1 opposite S4 or next to it). I recall that the distributed CCP4
dictionaries have different atom naming than the PDB chemical components
dictionary.  If this is the problem is that the naming is different this
should result
in large restraint violations that should be appear in the REFMAC output
(others can tell you where).

In general diffraction from Fe S clusters is so strong that the atom
positions
are pretty much determined from the electron density. But in case it helps
I did some work data mining restraints for Fe2S2 and Fe4S4 from CSD
structures (this has to be done manually). Please find attached the
resulting FES.cif and SF4.cif restraint dictionaries (distributed with
BUSTER).

Good luck,

Oliver
---
Dr Oliver Smart
Director SmartSci Limited http://www.smartsci.uk/
 Consultant Global Phasing Ltd http://www.globalphasing.com/

on 10/10/14 5:33 PM, Robbie Joosten robbie_joos...@hotmail.com wrote:

 Dear Stephen,

 The dictionary is very specific about atom names. If you have them swapped
 (as many PDB entries do). The angle and chiral volume restraints will
wreck
 your cluster. You also need to make sure to provide LINK records to attach
 the cluster to the surrounding cysteines.

 HTH,
 Robbie

 Sent from my Windows Phone
 
 Van: Stephen Carr
 Verzonden: 10-10-2014 18:07
 Aan: CCP4BB@JISCMAIL.AC.UK
 Onderwerp: [ccp4bb] Refinement of Iron-sulphur clusters

 Dear CCP4 BBers,

 I am currently refining a model of a protein containing three iron sulphur
 clusters using Refmac (v5.8.0078) and am getting some unusual results.
One
 of the clusters (3fe 4s) seems to behave resonably and the refined
electron
 density looks very nice.  The atoms in the others clusters (4Fe4S and
 4Fe3S), however, migrate towards the centre of the cluster producing Fo-Fc
 difference maps with strong negative density at the centre surrounded by
 blobs of positive density where the atoms should be.

 I have checkedthe refmac dictionaries and there are entries for each of
the
 clusters, so I shouldn't have to explicitly include a cif file for the
 clusters as an input right?  In fact, when I did try to include a
dictionary
 file for one of the clusters refmac gave a warning message in the log
about
 duplicate monomers dictionaries and it made no difference to the
 refinement/maps anyway.

 On checking the refmac log file there is no specific mention that there
are
 hetero-groups in the pdb file and no explicit mention that it is using any
 dictionaries during the refinement.  How can I tell if refmac is
 reading/using the dictionaries?  Also bonds within the clusters are
flagged
 up as outliers and deviating by more than 10 sigma from ideal suggesting
 that they are not getting read?

 I realise that something chemically odd could be going on at the centres,
 but if I omit them from the model and calculate maps very strong density
 comes back around where the atoms sat before refinement, so I am pretty
 confident that they are in the right place to start with.

 Any help with this problem would be greatly appreciated, I'm sure there is
 something obvious that I am missing but can't see what that is at the
 moment.  It is particularly confusing since one cluster apparently behaves
 while the others do not.  I could, of course, try phenix or buster, but I
 would like to get to the bottom of the problem with refmac if possible.

 Thanks very much in advance,

 Steve

 Dr Stephen Carr
 Research Complex at Harwell (RCaH)
 Rutherford Appleton Laboratory
 Harwell Oxford
 Didcot
 Oxon OX11 0FA
 United Kingdom
 Email stephen.c...@rc-harwell.ac.uk
 tel 01235 567717
 This email and any attachments may contain confidential, copyright and or
 privileged material, and are for the use

Re: [ccp4bb] Refinement of Iron-sulphur clusters

[Pedro Matias]

Deat CCP4ers,

In relation to this topic, I'd like to mention that SF4 has replaced FS4
as the Fe4S4 monomer in the CCP4 monomer library.

However, the dictionary values for bond lengths and angles are not
correct, and this is especially noticeable when the dictionary is used
in a high-resolution refinement (e.g., 1.05 A). In particular, the
Fe-S-Fe angles are all set to 90 degrees, when in fact some are smaller
and others are larger.

This introduces a bias in the final rms angle deviation values. Fe4S4 is
a distorted cubane, not a perfect cube.

Best regards,

Pedro Matias

Em 13-10-2014 12:09, Oliver Smart escreveu:
 Stephen,

 Robbie advice is 100% correct. Be careful about the naming of the sulphur
 atoms (is S1 opposite S4 or next to it). I recall that the distributed CCP4
 dictionaries have different atom naming than the PDB chemical components
 dictionary.  If this is the problem is that the naming is different this
 should result
 in large restraint violations that should be appear in the REFMAC output
 (others can tell you where).

 In general diffraction from Fe S clusters is so strong that the atom
 positions
 are pretty much determined from the electron density. But in case it helps
 I did some work data mining restraints for Fe2S2 and Fe4S4 from CSD
 structures (this has to be done manually). Please find attached the
 resulting FES.cif and SF4.cif restraint dictionaries (distributed with
 BUSTER).

 Good luck,

 Oliver
 ---
 Dr Oliver Smart
 Director SmartSci Limited http://www.smartsci.uk/
  Consultant Global Phasing Ltd http://www.globalphasing.com/

 on 10/10/14 5:33 PM, Robbie Joosten robbie_joos...@hotmail.com wrote:

 Dear Stephen,

 The dictionary is very specific about atom names. If you have them swapped
 (as many PDB entries do). The angle and chiral volume restraints will
 wreck
 your cluster. You also need to make sure to provide LINK records to attach
 the cluster to the surrounding cysteines.

 HTH,
 Robbie

 Sent from my Windows Phone
 
 Van: Stephen Carr
 Verzonden: 10-10-2014 18:07
 Aan: CCP4BB@JISCMAIL.AC.UK
 Onderwerp: [ccp4bb] Refinement of Iron-sulphur clusters

 Dear CCP4 BBers,

 I am currently refining a model of a protein containing three iron sulphur
 clusters using Refmac (v5.8.0078) and am getting some unusual results.
 One
 of the clusters (3fe 4s) seems to behave resonably and the refined
 electron
 density looks very nice.  The atoms in the others clusters (4Fe4S and
 4Fe3S), however, migrate towards the centre of the cluster producing Fo-Fc
 difference maps with strong negative density at the centre surrounded by
 blobs of positive density where the atoms should be.

 I have checkedthe refmac dictionaries and there are entries for each of
 the
 clusters, so I shouldn't have to explicitly include a cif file for the
 clusters as an input right?  In fact, when I did try to include a
 dictionary
 file for one of the clusters refmac gave a warning message in the log
 about
 duplicate monomers dictionaries and it made no difference to the
 refinement/maps anyway.

 On checking the refmac log file there is no specific mention that there
 are
 hetero-groups in the pdb file and no explicit mention that it is using any
 dictionaries during the refinement.  How can I tell if refmac is
 reading/using the dictionaries?  Also bonds within the clusters are
 flagged
 up as outliers and deviating by more than 10 sigma from ideal suggesting
 that they are not getting read?

 I realise that something chemically odd could be going on at the centres,
 but if I omit them from the model and calculate maps very strong density
 comes back around where the atoms sat before refinement, so I am pretty
 confident that they are in the right place to start with.

 Any help with this problem would be greatly appreciated, I'm sure there is
 something obvious that I am missing but can't see what that is at the
 moment.  It is particularly confusing since one cluster apparently behaves
 while the others do not.  I could, of course, try phenix or buster, but I
 would like to get to the bottom of the problem with refmac if possible.

 Thanks very much in advance,

 Steve

 Dr Stephen Carr
 Research Complex at Harwell (RCaH)
 Rutherford Appleton Laboratory
 Harwell Oxford
 Didcot
 Oxon OX11 0FA
 United Kingdom
 Email stephen.c...@rc-harwell.ac.uk
 tel 01235 567717
 This email and any attachments may contain confidential, copyright and or
 privileged material, and are for the use of the intended addressee only.
 If
 you are not the intended addressee or an authorized recipient of the
 addressee, please notify us of receipt by returning the e-mail and do not
 use, copy, retain, distribute or disclose the information in or attached
 to
 this email.

 Any views or opinions presented are solely those of the author and do not
 necessarily represent those of the Research Complex at Harwell.

 There is no guarantee that this email or any attachments are free 

Re: [ccp4bb] Refinement of Iron-sulphur clusters

[Robbie Joosten]

Dear Stephen,

The dictionary is very specific about atom names. If you have them swapped (as 
many PDB entries do). The angle and chiral volume restraints will wreck your 
cluster. You also need to make sure to provide LINK records to attach the 
cluster to the surrounding cysteines.

HTH,
Robbie

Sent from my Windows Phone

Van: Stephen Carr
Verzonden: 10-10-2014 18:07
Aan: CCP4BB@JISCMAIL.AC.UK
Onderwerp: [ccp4bb] Refinement of Iron-sulphur clusters

Dear CCP4 BBers,

I am currently refining a model of a protein containing three iron sulphur 
clusters using Refmac (v5.8.0078) and am getting some unusual results.  One of 
the clusters (3fe 4s) seems to behave resonably and the refined electron 
density looks very nice.  The atoms in the others clusters (4Fe4S and 4Fe3S), 
however, migrate towards the centre of the cluster producing Fo-Fc difference 
maps with strong negative density at the centre surrounded by blobs of positive 
density where the atoms should be.

I have checkedthe refmac dictionaries and there are entries for each of the 
clusters, so I shouldn't have to explicitly include a cif file for the clusters 
as an input right?  In fact, when I did try to include a dictionary file for 
one of the clusters refmac gave a warning message in the log about duplicate 
monomers dictionaries and it made no difference to the refinement/maps anyway.

On checking the refmac log file there is no specific mention that there are 
hetero-groups in the pdb file and no explicit mention that it is using any 
dictionaries during the refinement.  How can I tell if refmac is reading/using 
the dictionaries?  Also bonds within the clusters are flagged up as outliers 
and deviating by more than 10 sigma from ideal suggesting that they are not 
getting read?

I realise that something chemically odd could be going on at the centres, but 
if I omit them from the model and calculate maps very strong density comes back 
around where the atoms sat before refinement, so I am pretty confident that 
they are in the right place to start with.

Any help with this problem would be greatly appreciated, I'm sure there is 
something obvious that I am missing but can't see what that is at the moment.  
It is particularly confusing since one cluster apparently behaves while the 
others do not.  I could, of course, try phenix or buster, but I would like to 
get to the bottom of the problem with refmac if possible.

Thanks very much in advance,

Steve

Dr Stephen Carr
Research Complex at Harwell (RCaH)
Rutherford Appleton Laboratory
Harwell Oxford
Didcot
Oxon OX11 0FA
United Kingdom
Email stephen.c...@rc-harwell.ac.uk
tel 01235 567717
This email and any attachments may contain confidential, copyright and or 
privileged material, and are for the use of the intended addressee only. If you 
are not the intended addressee or an authorized recipient of the addressee, 
please notify us of receipt by returning the e-mail and do not use, copy, 
retain, distribute or disclose the information in or attached to this email.

Any views or opinions presented are solely those of the author and do not 
necessarily represent those of the Research Complex at Harwell.

There is no guarantee that this email or any attachments are free from viruses 
and we cannot accept liability for any damage which you may sustain as a result 
of software viruses which may be transmitted in or with the message.

We use an electronic filing system. Please send electronic versions of 
documents, unless paper is specifically requested.

This email may have a protective marking, for an explanation, please see:
http://www.mrc.ac.uk/About/informationandstandards/documentmarking/index.htm.

Re: [ccp4bb] Refinement problem

[Eleanor Dodson]

Rather hard to advise!

Options are:
 a)  The helical domain is wrongly positioned!
  b)  There are scaling problems or swathes of missing data which can
depress some parts of the electron density. If you use coot and reduce the
contour level in this region, is anything sensible visible?

A useful test is to set the occupancy of some bulky residues to 0.00,
repeat the refinement, then see whether you can see the missing atoms at
some level in the map.
  Eleanor





On 10 September 2014 03:20, Appu kumar appu.kum...@gmail.com wrote:

 Hello All,

 I am new to low resolution refinement parametrization and regularization.
 Crystal diffracted with high anisotropy reaching to 3.5A in one direction
 and 4.5A other direction. I am refining a structure at 3.9A resolution.
 Protein has two domain connected trhough a linker and is packed as tetramer
 in ASU. Refinement in phenix as well as in refmac leads to wipe away of
 electron density in helical domain of protein leaving only blobs of
 electron density while other domain have good amount of density after
 refinement. I need your precious and valuable suggestion for proceeding
 with refinement.

 Thanks in advance for your suggestion.

 Thank you

Re: [ccp4bb] Refinement with new ligands to PDB

[Danilo Belviso]

Dear Meisam,

In the past I had similar problems, because of jligand and other cif 
generators can make errors when generate cif files, as it has been 
written before. I have solved these problems by using sketcher (ccp4) 
and PRODRG server (http://davapc1.bioch.dundee.ac.uk/cgi-bin/prodrg).


I use the first one to create the pdb file (it is very easy to use) and 
the second to generate the restrains file both for refmac and for 
phenix.refine.


I hope I've helped you.

Danilo

Il 2014-04-01 01:21 Meisam Nosrati ha scritto:

Dear CCP4ers

I have crystallized a protein with a series of ligands that are not in 
the PDB.


I have made the pdb and the torsion libraries in the jligand and given
them the new three letter codes, and then I try the refinement in the
PHENIX or CCP4, but since these ligands are not in the library the
refinement aborts. Even when I open the ligand files in COOT and I
import the cif dictionary, still it does not recognize it, and does
not let me rotate around the bonds.

I need to know how to fix this problem, and how can I choose the three
letter codes for my ligands that are not already chosen by other
people.

Thanks in advance for your help

Meisam

Re: [ccp4bb] Refinement with new ligands to PDB

[meisam nosrati]

Thank you very much everyone for your comments.

I will try your ideas and try to get this thing to work.

I appreciate your help

Meisam


On Mon, Mar 31, 2014 at 7:21 PM, Meisam Nosrati meisam.nosr...@gmail.comwrote:

 Dear CCP4ers

 I have crystallized a protein with a series of ligands that are not in the
 PDB.

 I have made the pdb and the torsion libraries in the jligand and given
 them the new three letter codes, and then I try the refinement in the
 PHENIX or CCP4, but since these ligands are not in the library the
 refinement aborts. Even when I open the ligand files in COOT and I import
 the cif dictionary, still it does not recognize it, and does not let me
 rotate around the bonds.

 I need to know how to fix this problem, and how can I choose the three
 letter codes for my ligands that are not already chosen by other people.

 Thanks in advance for your help

 Meisam

Re: [ccp4bb] Refinement of data with pseudo translation symmetry

[Eleanor Dodson]

Well - your R values will probably appear higher than normal  - there
will be zones where all reflections are  weak..
but the maximum likelihood targets are meant to deal with this reasonably well.

It seems to work and the maps usually look OK! Eleanor



On 25 November 2013 22:31, Niu Tou niutou2...@gmail.com wrote:
 Dear All,

 Does any body know if the existence of pseudo translation symmetry will
 affect refinement ? If it does, is there any keyword or method to avoid it?
 Thanks!

 Best,
 Niu

Re: [ccp4bb] Refinement of crystals containing a mixture in the asymmetric unit

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Paul,

have you actually tried using the 'alternate location indicator' with
two different residues? I would not be surprised if that would work
with refmac.

Best,
Tim

On 08/23/2013 05:39 PM, Paul Paukstelis wrote:
 Greetings,
 
 We have been working on a few DNA crystals in which the asymmetric
 unit contains a stoichiometric (or nearly so) mixture of two
 similar but distinct oligonucleotides. The resolution is medium to
 low (2.7-2.8) but for a few of these there are some hints from the
 density for two different bases at the same position. I'm curious
 what the best way to approach refinement would be in this case.
 Alternate conformation doesn't really work since the residues have
 different nucleobases. Having two complete chains with 0.5
 occupancy is overkill since there are only 2 (or 4) positions in
 which the sequence differs. I tried just adding a second chain for
 the varying residues at 0.5 occupancy and adding link records to
 the original chain, however this doesn't seem to respect geometry
 of the phosphodiester for the flanking residues. I would appreciate
 suggestions or any examples in the PDB that might set me in the
 right direction.
 
 --paul
 

- -- 
- --
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A

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ite155+O8JylmpSS454gYXM=
=3lH/
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Re: [ccp4bb] Refinement of crystals containing a mixture in the asymmetric unit

[Pavel Afonine]

Hi Paul,
I would have them both in PDB file with different non-blanc altLocs and
arbitrary starting occupancies and that will work in refinement (in
phenix.refine for sure, can't tell for other programs).
Pavel



On Fri, Aug 23, 2013 at 8:39 AM, Paul Paukstelis
shocksofmig...@gmail.comwrote:

 Greetings,

 We have been working on a few DNA crystals in which the asymmetric unit
 contains a stoichiometric (or nearly so) mixture of two similar but
 distinct oligonucleotides. The resolution is medium to low (2.7-2.8) but
 for a few of these there are some hints from the density for two different
 bases at the same position. I'm curious what the best way to approach
 refinement would be in this case. Alternate conformation doesn't really
 work since the residues have different nucleobases. Having two complete
 chains with 0.5 occupancy is overkill since there are only 2 (or 4)
 positions in which the sequence differs. I tried just adding a second chain
 for the varying residues at 0.5 occupancy and adding link records to the
 original chain, however this doesn't seem to respect geometry of the
 phosphodiester for the flanking residues. I would appreciate suggestions or
 any examples in the PDB that might set me in the right direction.

 --paul

Re: [ccp4bb] Refinement of crystals containing a mixture in the asymmetric unit

[Paul Paukstelis]

As an update, this approach did not work in Refmac, but as Pavel 
suggested it worked fine with phenix.refine.


--paul

On 08/23/2013 11:51 AM, Tim Gruene wrote:

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Paul,

have you actually tried using the 'alternate location indicator' with
two different residues? I would not be surprised if that would work
with refmac.

Best,
Tim

On 08/23/2013 05:39 PM, Paul Paukstelis wrote:

Greetings,

We have been working on a few DNA crystals in which the asymmetric
unit contains a stoichiometric (or nearly so) mixture of two
similar but distinct oligonucleotides. The resolution is medium to
low (2.7-2.8) but for a few of these there are some hints from the
density for two different bases at the same position. I'm curious
what the best way to approach refinement would be in this case.
Alternate conformation doesn't really work since the residues have
different nucleobases. Having two complete chains with 0.5
occupancy is overkill since there are only 2 (or 4) positions in
which the sequence differs. I tried just adding a second chain for
the varying residues at 0.5 occupancy and adding link records to
the original chain, however this doesn't seem to respect geometry
of the phosphodiester for the flanking residues. I would appreciate
suggestions or any examples in the PDB that might set me in the
right direction.

--paul

- -- 
- --

Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A

-BEGIN PGP SIGNATURE-
Version: GnuPG v1.4.14 (GNU/Linux)
Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/

iD8DBQFSF4T7UxlJ7aRr7hoRAtC9AKCeIcRnKeCrsW4/QY7ad5xooRw73wCgvEpw
ite155+O8JylmpSS454gYXM=
=3lH/
-END PGP SIGNATURE-

Re: [ccp4bb] Refinement of partly occupied water molecules

[Eleanor Dodson]

You are desribing the reason many people limit their refinement to lower
resolution! I think it is probably universally true that there are multiple
conformations for sidechain/water networks at the surface, which we just
dont model properly.  If you are going to tackle the fine details you need
to judge how the nearby side chains are fitted as well as the water. And of
course then you can probably also see stuff present in the crystallisation
media.

The rewards are a lower R factor, and a better understanding of mobility I
guess.
Eleanor



On 12 July 2013 09:08, Stefan Krimmer krim...@staff.uni-marburg.de wrote:

 Dear all,

 I have a question concerning the refinement of partly occupied water
 molecules:

 in some of my macromolecular crystal structures with resolutions between
 1.1 - 1.4 Å, several round positive Fo-Fc electron density blobs are
 detectable which show after assignment of a water molecule to these blobs
 and subsequent refinement with Phenix.refine a good-looking  2Fo-Fc
 electron density. However, there also occurs a small negative Fo-Fc
 electron density detectable inside the 2Fo-Fc density blob. The negative
 Fo-Fc electron density disappears if the occupancy of the water molecule is
 automatically refined by Phenix.refine (occupancy manually set to a value
 below 100% followed by refinement) or manually set to 50% and fixed for
 this value (Fix occupancy option in phenix.refine). Therefore, I think
 these positions are partly occupied by water molecules, but I am not sure
 how I should handle it/how it is generally handled. Which one of the two
 options described above is the better one? I would be thankful for any
 advice and/or literature about this topic.

 Thank you for your help!

 Stefan

Re: [ccp4bb] Refinement of partly occupied water molecules

[Nat Echols]

On Fri, Jul 12, 2013 at 1:08 AM, Stefan Krimmer 
krim...@staff.uni-marburg.de wrote:

 in some of my macromolecular crystal structures with resolutions between
 1.1 - 1.4 Å, several round positive Fo-Fc electron density blobs are
 detectable which show after assignment of a water molecule to these blobs
 and subsequent refinement with Phenix.refine a good-looking  2Fo-Fc
 electron density. However, there also occurs a small negative Fo-Fc
 electron density detectable inside the 2Fo-Fc density blob. The negative
 Fo-Fc electron density disappears if the occupancy of the water molecule is
 automatically refined by Phenix.refine (occupancy manually set to a value
 below 100% followed by refinement) or manually set to 50% and fixed for
 this value (Fix occupancy option in phenix.refine). Therefore, I think
 these positions are partly occupied by water molecules, but I am not sure
 how I should handle it/how it is generally handled. Which one of the two
 options described above is the better one? I would be thankful for any
 advice and/or literature about this topic.


When I had to deal with this in the past, I followed this advice (from
Thomas Schneider):

http://www.embl-hamburg.de/~tschneider/shelxl/shelxl_faq/shelxlfaq.html#Q16

This is especially true at the resolutions you're working with; even with
subatomic resolution data I believe that the observation in the FAQ (that
refining the occupancies doesn't improve R-factors and may even make them
worse) will be true in most cases - and regardless of program used, btw.
(I can't remember if I ever tried comparing the outcomes myself, though.)

-Nat

Re: [ccp4bb] Refinement against frames

[Loes Kroon-Batenburg]

Dear Boaz,

Indeed, small molecule crystallographers are routinely converting pixels 
into I's and can refine structures to very low R-values, but only to a 
limited resolution. The Bragg intensities are very strong, and 
background scattering stays almost unnoticed. Once they start studying 
accurate electron densities the flaws in the models (Icalc) become 
apparent.
However, protein crystals are different: they have large disordered 
solvent regions, disorder in the proteins conformations, and background 
scattering of the mother liquor/air/crystal mount that may be even 
stronger than the many weak intensities. The disorder of the protein 
will lead to incoherent scattering that also produces significant 
background scattering, which at moderate B-factors  may make up half of 
the total scattering.  Converting pixel intensities into I_bragg (after 
subtracting some background) and refining against those (or F's) is 
clearly a simplification, and only gives us the average structure and 
not the true structure. The disorder may also lead to more structured 
non-Bragg scattering, which we call diffuse scattering, indicating that 
our crystal is in fact not periodic. Understanding what is really going 
on in our crystal, and trying to model the observed raw diffraction 
patterns is in fact very interesting, may solve the problems of trying 
to convert I's to F's, may give a better estimate of the 'average' 
structures and tell us how the protein molecules are really behaving (in 
the crystal).
Trying to model diffraction images comes with lots of additional 
problems, because instrumental characteristics have also to be modeled. 
However, it is a very interesting route to go.
There may be a moment in future where we think we can do this. It would 
be good if than we would have raw images available of all those weirdly 
diffracting crystals, that we managed in some way or another to extract 
I_bragg (or Ispot-Iback) from.


Greetings,
Loes.

On 06/24/13 14:21, Boaz Shaanan wrote:

Hi Tim,

I agree with you.  Another point to remember about this issue of pixel-F's  
(or I's) conversion is that small molecule crystallographers take the same route 
and produce structures with 1-2% R-factors, so this conversion is hardly our 
problem. The main culprit in the issues that have been discussed so lucidly on the 
BB recently have mostly to do with the vast amount of weak reflections in 
diffraction patterns of macromolecules (and how to decide on resolution in such 
situations). Digging into the peak/background pixels and signal/noise ratio there 
is just going to open another Pandora box.

My 2p thoughts.

  Cheers,

  Boaz


Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Israel

E-mail: bshaa...@bgu.ac.il
Phone: 972-8-647-2220  Skype: boaz.shaanan
Fax:   972-8-647-2992 or 972-8-646-1710






From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Tim Gruene 
[t...@shelx.uni-ac.gwdg.de]
Sent: Monday, June 24, 2013 2:59 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Refinement against frames

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear John,

actually I am not a friend of this idea. Processing software make an
excellent job of removing the instrumental part from our data. If we
start to integrate against frames, the next structural title might be
something like Crystal structure of ABC a xA resolution measured at
beamline xyz with a frame width of f degrees and a total rotation
range of phi degreees... the point I am trying to make: once
integrating against frames one may have to take a lot of issues into
account for interpreting the structure.
And do you think that refining against frames will actually give
greater chemical or biological insight into the sample, or will it
only give a more accurate description of the crystal contents? These
are two different things and the latter is - in my opinion - not what
structures are about.

Best, Tim

P.S.: I changed the subject line, because the thread based sorting of
my emails is soon going to exceed the width of my screem for the
original one.

On 06/24/2013 08:13 AM, Jrh wrote:

Dear Tom, I find this suggestion of using the full images an
excellent and visionary one. So, how to implement it? We are part
way along the path with James Holton's reverse Mosflm. The computer
memory challenge could be ameliorated by simple pixel averaging at
least initially. The diffuse scattering would be the ultimate gold
at the end of the rainbow. Peter Moore's new book, inter alia,
carries many splendid insights into the diffuse scattering in our
diffraction patterns. Fullprof analyses have become a firm trend in
other fields, admittedly with simpler computing overheads.
Greetings, John

Prof John R Helliwell DSc FInstP



On 21 Jun 2013, at 23:16, Terwilliger, Thomas C
terwilli...@lanl.gov  wrote:


I hope I am not duplicating too much of this fascinating

Re: [ccp4bb] Refinement against frames

[Boaz Shaanan]

Dear Loes,

Thanks for the message. To the best of my recollection (I actually come from 
small molecules crystallography) the problems of small molecule 
crystallographers when it comes to studying accurate e.d.'s (e.g. bond 
densities and such) have mostly to do with separating the effect of atomic 
thermal motion and true residual bond densities, i.e. mostly issues of 
modelling the thermal motion. TDS is a pain for small molecule crystallography 
and protein crystallographers. It's reminiscent of  the British weather - 
everybody complains about it but nobody does anything about it. Do small 
molecule crystallographers model TDS properly and correct the data for it 
nowadays in studies of accurate e.d.?

Modelling the thermal motion in proteins by B-factors is known to be a gross 
over-simplification because of many reasons, some of which you mentioned. TDS 
is another issue. There have been attempts in the past by several groups to 
deal with TDS in protein crystals but I'm not sure the community was convinced 
that it lead to improvement of the data. Whether TDS is the main culprit for 
the relatively high R factor of protein structures (that is relative to small 
molecules) is not clear. Modelling TDS (both the parts that arise from protein 
dynamics and crystal disorder) in protein data,  in order to improve our data 
and the resulting atomic models is a good thing.  Why should that logically 
lead to refinement against frames once the TDS has been modelled properly and 
the data corrected accordingly (future tense should be used here, actually), is 
not clear to me. I would think that working on one (or a few) data sets that 
suffer from severe TDS, correcting the data, and re-refining the models to see 
what difference it makes would be a good starting point. 

   Cheers,

   Boaz


Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Israel

E-mail: bshaa...@bgu.ac.il
Phone: 972-8-647-2220  Skype: boaz.shaanan
Fax:   972-8-647-2992 or 972-8-646-1710






From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Loes 
Kroon-Batenburg [l.m.j.kroon-batenb...@uu.nl]
Sent: Tuesday, June 25, 2013 1:09 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Refinement against frames

Dear Boaz,

Indeed, small molecule crystallographers are routinely converting pixels
into I's and can refine structures to very low R-values, but only to a
limited resolution. The Bragg intensities are very strong, and
background scattering stays almost unnoticed. Once they start studying
accurate electron densities the flaws in the models (Icalc) become
apparent.
However, protein crystals are different: they have large disordered
solvent regions, disorder in the proteins conformations, and background
scattering of the mother liquor/air/crystal mount that may be even
stronger than the many weak intensities. The disorder of the protein
will lead to incoherent scattering that also produces significant
background scattering, which at moderate B-factors  may make up half of
the total scattering.  Converting pixel intensities into I_bragg (after
subtracting some background) and refining against those (or F's) is
clearly a simplification, and only gives us the average structure and
not the true structure. The disorder may also lead to more structured
non-Bragg scattering, which we call diffuse scattering, indicating that
our crystal is in fact not periodic. Understanding what is really going
on in our crystal, and trying to model the observed raw diffraction
patterns is in fact very interesting, may solve the problems of trying
to convert I's to F's, may give a better estimate of the 'average'
structures and tell us how the protein molecules are really behaving (in
the crystal).
Trying to model diffraction images comes with lots of additional
problems, because instrumental characteristics have also to be modeled.
However, it is a very interesting route to go.
There may be a moment in future where we think we can do this. It would
be good if than we would have raw images available of all those weirdly
diffracting crystals, that we managed in some way or another to extract
I_bragg (or Ispot-Iback) from.

Greetings,
Loes.

On 06/24/13 14:21, Boaz Shaanan wrote:
 Hi Tim,

 I agree with you.  Another point to remember about this issue of pixel-F's  
 (or I's) conversion is that small molecule crystallographers take the same 
 route and produce structures with 1-2% R-factors, so this conversion is 
 hardly our problem. The main culprit in the issues that have been discussed 
 so lucidly on the BB recently have mostly to do with the vast amount of weak 
 reflections in diffraction patterns of macromolecules (and how to decide on 
 resolution in such situations). Digging into the peak/background pixels and 
 signal/noise ratio there is just going to open another Pandora box.

 My 2p thoughts.

   Cheers,

   Boaz

Re: [ccp4bb] Refinement against frames

[Loes Kroon-Batenburg]

Dear Boaz,


Dear Boaz,
On 06/25/13 14:09, Boaz Shaanan wrote:

Dear Loes,

Thanks for the message. To the best of my recollection (I actually come from 
small molecules crystallography) the problems of small molecule 
crystallographers when it comes to studying accurate e.d.'s (e.g. bond 
densities and such) have mostly to do with separating the effect of atomic 
thermal motion and true residual bond densities, i.e. mostly issues of 
modelling the thermal motion. TDS is a pain for small molecule crystallography 
and protein crystallographers. It's reminiscent of  the British weather - 
everybody complains about it but nobody does anything about it. Do small 
molecule crystallographers model TDS properly and correct the data for it 
nowadays in studies of accurate e.d.
I agree that in small molecule crystallography  thermal motion has to be 
modelled accurately, certainly for accurate electron density studies. 
The thermal motion leads to TDS, and can be found at /near Bragg 
positions. This is mostly ignored.

Modelling the thermal motion in proteins by B-factors is known to be a gross 
over-simplification because of many reasons, some of which you mentioned. TDS 
is another issue. There have been attempts in the past by several groups to 
deal with TDS in protein crystals but I'm not sure the community was convinced 
that it lead to improvement of the data. Whether TDS is the main culprit for 
the relatively high R factor of protein structures (that is relative to small 
molecules) is not clear. Modelling TDS (both the parts that arise from protein 
dynamics and crystal disorder) in protein data,  in order to improve our data 
and the resulting atomic models is a good thing.
In proteins also TDS occurs but more importantly large (correlated) 
domain motions lead to scattering in between Bragg peaks, in the shape 
of large diffuse clouds or streaks or whatever.



Why should that logically lead to refinement against frames once the TDS has 
been modelled properly and the data corrected accordingly (future tense should 
be used here, actually), is not clear to me. I would think that working on one 
(or a few) data sets that suffer from severe TDS, correcting the data, and 
re-refining the models to see what difference it makes would be a good starting 
point.
The fact that these can be observed tells us that proteins crystals show 
much more dynamics (frozen in as static disorder) than we tend to 
assume. And thus our description of protein structures is simplified.


Best wishes,
Loes.

Cheers,

Boaz


Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Israel

E-mail: bshaa...@bgu.ac.il
Phone: 972-8-647-2220  Skype: boaz.shaanan
Fax:   972-8-647-2992 or 972-8-646-1710






From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Loes 
Kroon-Batenburg [l.m.j.kroon-batenb...@uu.nl]
Sent: Tuesday, June 25, 2013 1:09 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Refinement against frames

Dear Boaz,

Indeed, small molecule crystallographers are routinely converting pixels
into I's and can refine structures to very low R-values, but only to a
limited resolution. The Bragg intensities are very strong, and
background scattering stays almost unnoticed. Once they start studying
accurate electron densities the flaws in the models (Icalc) become
apparent.
However, protein crystals are different: they have large disordered
solvent regions, disorder in the proteins conformations, and background
scattering of the mother liquor/air/crystal mount that may be even
stronger than the many weak intensities. The disorder of the protein
will lead to incoherent scattering that also produces significant
background scattering, which at moderate B-factors  may make up half of
the total scattering.  Converting pixel intensities into I_bragg (after
subtracting some background) and refining against those (or F's) is
clearly a simplification, and only gives us the average structure and
not the true structure. The disorder may also lead to more structured
non-Bragg scattering, which we call diffuse scattering, indicating that
our crystal is in fact not periodic. Understanding what is really going
on in our crystal, and trying to model the observed raw diffraction
patterns is in fact very interesting, may solve the problems of trying
to convert I's to F's, may give a better estimate of the 'average'
structures and tell us how the protein molecules are really behaving (in
the crystal).
Trying to model diffraction images comes with lots of additional
problems, because instrumental characteristics have also to be modeled.
However, it is a very interesting route to go.
There may be a moment in future where we think we can do this. It would
be good if than we would have raw images available of all those weirdly
diffracting crystals, that we managed in some way or another to extract
I_bragg (or Ispot-Iback) from.

Greetings

Re: [ccp4bb] Refinement against frames

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear John,

actually I am not a friend of this idea. Processing software make an
excellent job of removing the instrumental part from our data. If we
start to integrate against frames, the next structural title might be
something like Crystal structure of ABC a xA resolution measured at
beamline xyz with a frame width of f degrees and a total rotation
range of phi degreees... the point I am trying to make: once
integrating against frames one may have to take a lot of issues into
account for interpreting the structure.
And do you think that refining against frames will actually give
greater chemical or biological insight into the sample, or will it
only give a more accurate description of the crystal contents? These
are two different things and the latter is - in my opinion - not what
structures are about.

Best, Tim

P.S.: I changed the subject line, because the thread based sorting of
my emails is soon going to exceed the width of my screem for the
original one.

On 06/24/2013 08:13 AM, Jrh wrote:
 Dear Tom, I find this suggestion of using the full images an
 excellent and visionary one. So, how to implement it? We are part
 way along the path with James Holton's reverse Mosflm. The computer
 memory challenge could be ameliorated by simple pixel averaging at
 least initially. The diffuse scattering would be the ultimate gold
 at the end of the rainbow. Peter Moore's new book, inter alia,
 carries many splendid insights into the diffuse scattering in our
 diffraction patterns. Fullprof analyses have become a firm trend in
 other fields, admittedly with simpler computing overheads. 
 Greetings, John
 
 Prof John R Helliwell DSc FInstP
 
 
 
 On 21 Jun 2013, at 23:16, Terwilliger, Thomas C
 terwilli...@lanl.gov wrote:
 
 I hope I am not duplicating too much of this fascinating
 discussion with these comments:  perhaps the main reason there is
 confusion about what to do is that neither F nor I is really the
 most suitable thing to use in refinement.  As pointed out several
 times in different ways, we don't measure F or I, we only measure
 counts on a detector.  As a convenience, we process our
 diffraction images to estimate I or F and their uncertainties and
 model these uncertainties as simple functions (e.g., a Gaussian).
 There is no need in principle to do that, and if we were to
 refine instead against the raw image data these issues about
 positivity would disappear and our structures might even be a
 little better.
 
 Our standard procedure is to estimate F or I from counts on the
 detector, then to use these estimates of F or I in refinement.
 This is not so easy to do right because F or I contain many terms
 coming from many pixels and it is hard to model their statistics
 in detail.  Further, attempts we make to estimate either F or I
 as physically plausible values (e.g., using the fact that they
 are not negative) will generally be biased (the values after
 correction will generally be systematically low or systematically
 high, as is true for the French and Wilson correction and as
 would be true for the truncation of I at zero or above).
 
 Randy's method for intensity refinement is an improvement because
 the statistics are treated more fully than just using an estimate
 of F or I and assuming its uncertainty has a simple distribution.
 So why not avoid all the problems with modeling the statistics of
 processed data and instead refine against the raw data.  From the
 structural model you calculate F, from F and a detailed model of
 the experiment (the same model that is currently used in data
 processing) you calculate the counts expected on each pixel. Then
 you calculate the likelihood of the data given your models of the
 structure and of the experiment.  This would have lots of
 benefits because it would allow improved descriptions of the
 experiment (decay, absorption, detector sensitivity, diffuse
 scattering and other background on the images,on and on)
 that could lead to more accurate structures in the end.  Of
 course there are some minor issues about putting all this in
 computer memory for refinement
 
 -Tom T  From: CCP4
 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Phil
 [p...@mrc-lmb.cam.ac.uk] Sent: Friday, June 21, 2013 2:50 PM To:
 CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] ctruncate bug?
 
 However you decide to argue the point, you must consider _all_
 the observations of a reflection (replicates and symmetry
 related) together when you infer Itrue or F etc, otherwise you
 will bias the result even more. Thus you cannot (easily) do it
 during integration
 
 Phil
 
 Sent from my iPad
 
 On 21 Jun 2013, at 20:30, Douglas Theobald
 dtheob...@brandeis.edu wrote:
 
 On Jun 21, 2013, at 2:48 PM, Ed Pozharski
 epozh...@umaryland.edu wrote:
 
 Douglas,
 Observed intensities are the best estimates that we can
 come up with in an experiment.
 I also agree with this, and this is 

Re: [ccp4bb] Refinement against frames

[Boaz Shaanan]

Hi Tim,

I agree with you.  Another point to remember about this issue of pixel-F's  
(or I's) conversion is that small molecule crystallographers take the same 
route and produce structures with 1-2% R-factors, so this conversion is hardly 
our problem. The main culprit in the issues that have been discussed so lucidly 
on the BB recently have mostly to do with the vast amount of weak reflections 
in diffraction patterns of macromolecules (and how to decide on resolution in 
such situations). Digging into the peak/background pixels and signal/noise 
ratio there is just going to open another Pandora box. 

My 2p thoughts.

 Cheers,

 Boaz 


Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Israel

E-mail: bshaa...@bgu.ac.il
Phone: 972-8-647-2220  Skype: boaz.shaanan
Fax:   972-8-647-2992 or 972-8-646-1710






From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Tim Gruene 
[t...@shelx.uni-ac.gwdg.de]
Sent: Monday, June 24, 2013 2:59 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Refinement against frames

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear John,

actually I am not a friend of this idea. Processing software make an
excellent job of removing the instrumental part from our data. If we
start to integrate against frames, the next structural title might be
something like Crystal structure of ABC a xA resolution measured at
beamline xyz with a frame width of f degrees and a total rotation
range of phi degreees... the point I am trying to make: once
integrating against frames one may have to take a lot of issues into
account for interpreting the structure.
And do you think that refining against frames will actually give
greater chemical or biological insight into the sample, or will it
only give a more accurate description of the crystal contents? These
are two different things and the latter is - in my opinion - not what
structures are about.

Best, Tim

P.S.: I changed the subject line, because the thread based sorting of
my emails is soon going to exceed the width of my screem for the
original one.

On 06/24/2013 08:13 AM, Jrh wrote:
 Dear Tom, I find this suggestion of using the full images an
 excellent and visionary one. So, how to implement it? We are part
 way along the path with James Holton's reverse Mosflm. The computer
 memory challenge could be ameliorated by simple pixel averaging at
 least initially. The diffuse scattering would be the ultimate gold
 at the end of the rainbow. Peter Moore's new book, inter alia,
 carries many splendid insights into the diffuse scattering in our
 diffraction patterns. Fullprof analyses have become a firm trend in
 other fields, admittedly with simpler computing overheads.
 Greetings, John

 Prof John R Helliwell DSc FInstP



 On 21 Jun 2013, at 23:16, Terwilliger, Thomas C
 terwilli...@lanl.gov wrote:

 I hope I am not duplicating too much of this fascinating
 discussion with these comments:  perhaps the main reason there is
 confusion about what to do is that neither F nor I is really the
 most suitable thing to use in refinement.  As pointed out several
 times in different ways, we don't measure F or I, we only measure
 counts on a detector.  As a convenience, we process our
 diffraction images to estimate I or F and their uncertainties and
 model these uncertainties as simple functions (e.g., a Gaussian).
 There is no need in principle to do that, and if we were to
 refine instead against the raw image data these issues about
 positivity would disappear and our structures might even be a
 little better.

 Our standard procedure is to estimate F or I from counts on the
 detector, then to use these estimates of F or I in refinement.
 This is not so easy to do right because F or I contain many terms
 coming from many pixels and it is hard to model their statistics
 in detail.  Further, attempts we make to estimate either F or I
 as physically plausible values (e.g., using the fact that they
 are not negative) will generally be biased (the values after
 correction will generally be systematically low or systematically
 high, as is true for the French and Wilson correction and as
 would be true for the truncation of I at zero or above).

 Randy's method for intensity refinement is an improvement because
 the statistics are treated more fully than just using an estimate
 of F or I and assuming its uncertainty has a simple distribution.
 So why not avoid all the problems with modeling the statistics of
 processed data and instead refine against the raw data.  From the
 structural model you calculate F, from F and a detailed model of
 the experiment (the same model that is currently used in data
 processing) you calculate the counts expected on each pixel. Then
 you calculate the likelihood of the data given your models of the
 structure and of the experiment.  This would have lots of
 benefits because it would allow improved

Re: [ccp4bb] Refinement against frames

[Mark J van Raaij]

Hi Tim,
I don't follow your point...frames are just data, and with more information 
than after integration. The data after integration is also to some extent 
dependent on the beamline.
It should indeed give a more accurate description of the crystal contents - 
whether that in turn will translate into greater chemical or biological insight 
(now or some time in the future) will depend on the specific case (and on the 
interpreter).
Mark
Mark J van Raaij
Lab 20B
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
c/Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://www.cnb.csic.es/~mjvanraaij





On 24 Jun 2013, at 13:59, Tim Gruene wrote:

 -BEGIN PGP SIGNED MESSAGE-
 Hash: SHA1
 
 Dear John,
 
 actually I am not a friend of this idea. Processing software make an
 excellent job of removing the instrumental part from our data. If we
 start to integrate against frames, the next structural title might be
 something like Crystal structure of ABC a xA resolution measured at
 beamline xyz with a frame width of f degrees and a total rotation
 range of phi degreees... the point I am trying to make: once
 integrating against frames one may have to take a lot of issues into
 account for interpreting the structure.
 And do you think that refining against frames will actually give
 greater chemical or biological insight into the sample, or will it
 only give a more accurate description of the crystal contents? These
 are two different things and the latter is - in my opinion - not what
 structures are about.
 
 Best, Tim
 
 P.S.: I changed the subject line, because the thread based sorting of
 my emails is soon going to exceed the width of my screem for the
 original one.
 
 On 06/24/2013 08:13 AM, Jrh wrote:
 Dear Tom, I find this suggestion of using the full images an
 excellent and visionary one. So, how to implement it? We are part
 way along the path with James Holton's reverse Mosflm. The computer
 memory challenge could be ameliorated by simple pixel averaging at
 least initially. The diffuse scattering would be the ultimate gold
 at the end of the rainbow. Peter Moore's new book, inter alia,
 carries many splendid insights into the diffuse scattering in our
 diffraction patterns. Fullprof analyses have become a firm trend in
 other fields, admittedly with simpler computing overheads. 
 Greetings, John
 
 Prof John R Helliwell DSc FInstP
 
 
 
 On 21 Jun 2013, at 23:16, Terwilliger, Thomas C
 terwilli...@lanl.gov wrote:
 
 I hope I am not duplicating too much of this fascinating
 discussion with these comments:  perhaps the main reason there is
 confusion about what to do is that neither F nor I is really the
 most suitable thing to use in refinement.  As pointed out several
 times in different ways, we don't measure F or I, we only measure
 counts on a detector.  As a convenience, we process our
 diffraction images to estimate I or F and their uncertainties and
 model these uncertainties as simple functions (e.g., a Gaussian).
 There is no need in principle to do that, and if we were to
 refine instead against the raw image data these issues about
 positivity would disappear and our structures might even be a
 little better.
 
 Our standard procedure is to estimate F or I from counts on the
 detector, then to use these estimates of F or I in refinement.
 This is not so easy to do right because F or I contain many terms
 coming from many pixels and it is hard to model their statistics
 in detail.  Further, attempts we make to estimate either F or I
 as physically plausible values (e.g., using the fact that they
 are not negative) will generally be biased (the values after
 correction will generally be systematically low or systematically
 high, as is true for the French and Wilson correction and as
 would be true for the truncation of I at zero or above).
 
 Randy's method for intensity refinement is an improvement because
 the statistics are treated more fully than just using an estimate
 of F or I and assuming its uncertainty has a simple distribution.
 So why not avoid all the problems with modeling the statistics of
 processed data and instead refine against the raw data.  From the
 structural model you calculate F, from F and a detailed model of
 the experiment (the same model that is currently used in data
 processing) you calculate the counts expected on each pixel. Then
 you calculate the likelihood of the data given your models of the
 structure and of the experiment.  This would have lots of
 benefits because it would allow improved descriptions of the
 experiment (decay, absorption, detector sensitivity, diffuse
 scattering and other background on the images,on and on)
 that could lead to more accurate structures in the end.  Of
 course there are some minor issues about putting all this in
 computer memory for refinement
 
 -Tom T  From: CCP4
 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Phil
 

Re: [ccp4bb] Refinement against frames

[Jrh]

Dear Tim,
With a full interpretation of the diffuse scattering as well how about papers 
becoming entitled:-
The structure and dynamics of enzyme X
As you intimate some diffuse scattering is crystal dependent ie phonons 
derived. Other aspects are however not correlated over multiple unit cells but 
thereby largely related to the dynamics of our macromolecules. (largely means 
we need to allow for static disorder and / or chemical variants possibilities). 

Re instrument aspects:-
The days of instrument setting dependent (eg due to varying magnetic fields as 
we changed xtod) detector response are indeed fortunately behind us. That said 
we might learn something on the instrument aspect doing things against the 
detector plane. Another aspect for example is detailed prediction of spot 
shape, although perhaps for the purists amongst us (eg see Greenhough, 
Helliwell and Rule 1983 JAC), but may add insights into I - I bg, ie the spot 
shape prior can be known. This can be done processing 'forwards or backwards'. 

Greetings,
John

Prof John R Helliwell DSc 
 
 

On 24 Jun 2013, at 12:59, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote:

 -BEGIN PGP SIGNED MESSAGE-
 Hash: SHA1
 
 Dear John,
 
 actually I am not a friend of this idea. Processing software make an
 excellent job of removing the instrumental part from our data. If we
 start to integrate against frames, the next structural title might be
 something like Crystal structure of ABC a xA resolution measured at
 beamline xyz with a frame width of f degrees and a total rotation
 range of phi degreees... the point I am trying to make: once
 integrating against frames one may have to take a lot of issues into
 account for interpreting the structure.
 And do you think that refining against frames will actually give
 greater chemical or biological insight into the sample, or will it
 only give a more accurate description of the crystal contents? These
 are two different things and the latter is - in my opinion - not what
 structures are about.
 
 Best, Tim
 
 P.S.: I changed the subject line, because the thread based sorting of
 my emails is soon going to exceed the width of my screem for the
 original one.
 
 On 06/24/2013 08:13 AM, Jrh wrote:
 Dear Tom, I find this suggestion of using the full images an
 excellent and visionary one. So, how to implement it? We are part
 way along the path with James Holton's reverse Mosflm. The computer
 memory challenge could be ameliorated by simple pixel averaging at
 least initially. The diffuse scattering would be the ultimate gold
 at the end of the rainbow. Peter Moore's new book, inter alia,
 carries many splendid insights into the diffuse scattering in our
 diffraction patterns. Fullprof analyses have become a firm trend in
 other fields, admittedly with simpler computing overheads. 
 Greetings, John
 
 Prof John R Helliwell DSc FInstP
 
 
 
 On 21 Jun 2013, at 23:16, Terwilliger, Thomas C
 terwilli...@lanl.gov wrote:
 
 I hope I am not duplicating too much of this fascinating
 discussion with these comments:  perhaps the main reason there is
 confusion about what to do is that neither F nor I is really the
 most suitable thing to use in refinement.  As pointed out several
 times in different ways, we don't measure F or I, we only measure
 counts on a detector.  As a convenience, we process our
 diffraction images to estimate I or F and their uncertainties and
 model these uncertainties as simple functions (e.g., a Gaussian).
 There is no need in principle to do that, and if we were to
 refine instead against the raw image data these issues about
 positivity would disappear and our structures might even be a
 little better.
 
 Our standard procedure is to estimate F or I from counts on the
 detector, then to use these estimates of F or I in refinement.
 This is not so easy to do right because F or I contain many terms
 coming from many pixels and it is hard to model their statistics
 in detail.  Further, attempts we make to estimate either F or I
 as physically plausible values (e.g., using the fact that they
 are not negative) will generally be biased (the values after
 correction will generally be systematically low or systematically
 high, as is true for the French and Wilson correction and as
 would be true for the truncation of I at zero or above).
 
 Randy's method for intensity refinement is an improvement because
 the statistics are treated more fully than just using an estimate
 of F or I and assuming its uncertainty has a simple distribution.
 So why not avoid all the problems with modeling the statistics of
 processed data and instead refine against the raw data.  From the
 structural model you calculate F, from F and a detailed model of
 the experiment (the same model that is currently used in data
 processing) you calculate the counts expected on each pixel. Then
 you calculate the likelihood of the data given your models of the
 structure and of the experiment.  This would have lots of
 benefits 

Re: [ccp4bb] refinement hanging--what am I missing?

[Eleanor Dodson]

What is the relationship between the molecules in the spacegroups - it
seems likely that in the bigger cell there is a NC translation of
~(1/2,y,z)?  ? (A2 ~ twice a1) That can be checked by the native
Patterson..

If so that gives a class of significantly weaker reflections for h odd, and
that usually does lead to higher R factors.
It also means that the h00 reflections will be weak for h odd, and that the
SG assignment could be either P2 2i2i  or P21 2i2i - you need to test
both..

However that doesnt explain the smaller R factors, unless the packing there
also gave rise to a NC translation..  Again checked from the native
Patterson..

Eleanor


On 29 April 2013 16:14, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote:

 -BEGIN PGP SIGNED MESSAGE-
 Hash: SHA1

 Hi Ed,

 just FYI: while this does not apply in the ccp4/mtz world, the fact
 you might be missing is that in some refinement environments merging
 is carried out by the refinement program so that the actual data can
 be kept unmerged and e.g. the information you refer to does not get
 lost (although scaling would smoothen it to a certain extent but at
 least does not erase it as mergin does). In that case you could expand
 to P1 (as far as I understand).

 best,
 Tim

 On 04/29/2013 04:13 PM, Edward A. Berry wrote:
  herman.schreu...@sanofi.com wrote:
  I would process or expand the data to P1, also expand your pdb
  file to P1 and refine in P1 to see what happens. I would also run
  Phaser or some other molecular replacement program on the P1 data
  to see what comes out.
 
 
  process or expand? I don't understand how there can really be a
  choice here. If the data is expanded from higher symmetry, it will
  precisely have that higher symmetry, i.e. all reflections that were
  equivalent in the higher symmetry will be identical. Any
  information about asymmetry was lost when the data were merged in
  the higher symmetry space group. Won't refinement by minimizing a
  target function have exactly the same solution?
 
  Or not exactly, because the refinement program can introduce
  asymmetry by allowing different phases for the equivalent
  reflections. But where is the information to generate that
  asymmetry coming from?
 
  There is probably something I'm missing here, but i would
  definitely reprocess in the lower symmetry if the rotation angle
  was sufficient to give good completeness.
 
  eab
 

 - --
 - --
 Dr Tim Gruene
 Institut fuer anorganische Chemie
 Tammannstr. 4
 D-37077 Goettingen

 GPG Key ID = A46BEE1A

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 Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/

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 =F/Om
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Re: [ccp4bb] refinement hanging--what am I missing?

[Herman . Schreuder]

Dear Patrick,

I don't know how many comments you got offline, but here are my comments:

1) Ice rings. Even rings that are hardly visible by eye can disturb the data 
such that Rfactors get stuck in the range you mention. Especially XDS, which is 
otherwise an excellent data processing program, performs poor in the presence 
of ice rings. You may want to look carefulla at your diffraction images, the 
wilson plot and at which resolutions you find most outlyers. 

2) Crystal packing. With 2 or 4 molecules in the asymmetric unit, a~b or 2a~b 
and space group P(2)(1)2(1)(2)(1), you have plenty possibilities for pseudo 
crystallographic symmetry. Since you have high resolution data, I would process 
or expand the data to P1, also expand your pdb file to P1 and refine in P1 to 
see what happens. I would also run Phaser or some other molecular replacement 
program on the P1 data to see what comes out.

Good luck!
Herman

 

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Patrick 
Loll
Sent: Saturday, April 27, 2013 12:32 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] refinement hanging--what am I missing?

Responding to a couple of questions from Ethan, Charlie, and Phil:

Ethan: Using the default bulk solvent modeling in Phenix; no selenium, but I'll 
double-check wavelengths as a sanity check for scattering factors (but several 
other native data sets from the same synchrotron trip refined beautifully, so I 
suspect there's no gross boo-boos of this nature...)

Charlie:  Solvent regions are pretty clean; I haven't tried any flipping (these 
are molecular replacement models, so it didn't occur to me...). I tried 
applying NCS in one case (the smaller cell) and it had no apparent effect on 
the refinement. The Fo-Fc map has no strong features crying out for 
interpretation. Just based on geometry and map appearance, I'd be inclined to 
say the refinement is done, were it not for the crappy R values.

Phil:  I used TLS for refinement in both xtal forms; it gives a small 
improvement (0.01-0.03) in Rfree in both cases, but nothing magic. I simply 
used one monomer/TLS group (these are ubiquitin variants, so the monomer itself 
is pretty much a little rock, without any internal domain motions). There are 
the usual complement of disordered side chains, but nothing unusual, and  98% 
of the main chain is accounted for. Haven't tried Arp/wArp yet...

Excellent thoughts, keep those cards and letters coming. I'm still chewing on 
the substantive comments from Dean and Adrian...

Thanks,

Pat

On 26 Apr 2013, at 6:17 PM, Carter, Charlie wrote:

 Hi Pat,
 
 Your stats aren't all that bad, but I share your discomfort. 
 
 Do the solvent regions retain any significant features? Have you tried 
 flipping those features? Have you applied NCS? What does the Fo - Fc map look 
 like?
 
 Charlie
 
 On Apr 26, 2013, at 5:38 PM, Patrick Loll wrote:
 
 Hi all,
 
 Here is a problem that's been annoying me, and demanding levels of thought 
 all out of proportion with the importance of the project:
 
 I have two related crystal forms of the same small protein. In both cases, 
 the data look quite decent, and extend beyond 2 A, but the refinement stalls 
 with statistics that are just bad enough to make me deeply uncomfortable. 
 However, the maps look pretty good, and there's no obvious path to push the 
 refinement further. Xtriage doesn't raise any red flags, nor does running 
 the data through the Yeates twinning server.
 
 Xtal form 1: P22(1)2(1), a=29.0, b=57.4, c=67.4; 2 molecules/AU. 
 Resolution of data ~ 1.9 Å. Refinement converges with R/Rfree = 
 0.24/0.27
 
 Xtal form 2: P2(1)2(1)2(1), a=59.50, b=61.1, c=67.2; 4 molecules/AU. 
 Resolution of data ~ 1.7 Å. Refinement converges w/ R/Rfree = 
 0.21/0.26
 
 As you would expect, the packing is essentially the same in both crystal 
 forms. 
 
 It's interesting to note (but is it relevant?) that the packing is quite 
 dense--solvent content is only 25-30%.
 
 This kind of stalling at high R values smells like a twin problem, but it's 
 not clear to me what specific kind of twinning might explain this behavior.
 
 Any thoughts about what I might be missing here?
 
 Thanks,
 
 Pat
 
 
 -
 --
 Patrick J. Loll, Ph. D.  
 Professor of Biochemistry  Molecular Biology Director, Biochemistry 
 Graduate Program Drexel University College of Medicine Room 10-102 
 New College Building
 245 N. 15th St., Mailstop 497
 Philadelphia, PA  19102-1192  USA
 
 (215) 762-7706
 pat.l...@drexelmed.edu
 

Re: [ccp4bb] refinement hanging--what am I missing?

[Edward A. Berry]

herman.schreu...@sanofi.com wrote:

I would process or expand the data to P1, also expand your pdb file to P1 and 
refine in P1 to see what happens. I would also run Phaser or some other 
molecular replacement program on the P1 data to see what comes out.



process or expand? I don't understand how there can really be a choice here.
If the data is expanded from higher symmetry, it will precisely have that
higher symmetry, i.e. all reflections that were equivalent in the higher
symmetry will be identical. Any information about asymmetry was lost when
the data were merged in the higher symmetry space group. Won't refinement
by minimizing a target function have exactly the same solution?

Or not exactly, because the refinement program can introduce asymmetry
by allowing different phases for the equivalent reflections.
But where is the information to generate that asymmetry coming from?

There is probably something I'm missing here, but i would definitely
reprocess in the lower symmetry if the rotation angle was sufficient to
give good completeness.

eab

Re: [ccp4bb] refinement hanging--what am I missing?

[Herman . Schreuder]

Hi Edward,

I and also others in the bulletin boards have had cases where the protein would 
build e.g. tetramers with internal 222 symmetry, which would pack with the 
2-folds parallel to the crystallographic 2-folds with say one 2-fold a little 
shifted from the crystallographic position. In these cases, the internal 222 
symmetry overwhelmed the contribution of the small shift and all processing 
programs, including Pointless would insist that the crystal was P2x2x2x. Also 
the Rfactors (Rsym, Rmeas) would be the same whether processing in P222 or P2.

However, the only way to pack the proteins correctly in the unit cell, would be 
to process in P2 or even in P1 and run molecular replacement in P2 or P1 and 
than reconstruct the most likely true space group, for which now the program 
Zanuda exists. Since in these cases the diffraction patterns would have 
perfect (within experimental error) P222 symmetry, I think it would be valid 
to expand the P222 data to P2 of P1. Of course collecting the full data set is 
still the best way to go.

The asymmetry is generated by the way the molecular replacement program places 
the molecules in the asymmetric unit and will, as you mention, only be in the 
phases. Expanding to P1 and running molecular replacement in P1 would test all 
possibilities at once, but the signal would be weaker. A more cautious approach 
would be to process in the three possible P2 spacegroups (choosing the 2-fold 
along a, b or c) and run molecular replacement for each option. It may also be 
that your problem lies elsewhere.

Best,
Herman
 

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Edward A. 
Berry
Sent: Monday, April 29, 2013 4:14 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] refinement hanging--what am I missing?

herman.schreu...@sanofi.com wrote:
 I would process or expand the data to P1, also expand your pdb file to P1 and 
 refine in P1 to see what happens. I would also run Phaser or some other 
 molecular replacement program on the P1 data to see what comes out.


process or expand? I don't understand how there can really be a choice here.
If the data is expanded from higher symmetry, it will precisely have that 
higher symmetry, i.e. all reflections that were equivalent in the higher 
symmetry will be identical. Any information about asymmetry was lost when the 
data were merged in the higher symmetry space group. Won't refinement by 
minimizing a target function have exactly the same solution?

Or not exactly, because the refinement program can introduce asymmetry by 
allowing different phases for the equivalent reflections.
But where is the information to generate that asymmetry coming from?

There is probably something I'm missing here, but i would definitely reprocess 
in the lower symmetry if the rotation angle was sufficient to give good 
completeness.

eab

Re: [ccp4bb] refinement hanging--what am I missing?

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Hi Ed,

just FYI: while this does not apply in the ccp4/mtz world, the fact
you might be missing is that in some refinement environments merging
is carried out by the refinement program so that the actual data can
be kept unmerged and e.g. the information you refer to does not get
lost (although scaling would smoothen it to a certain extent but at
least does not erase it as mergin does). In that case you could expand
to P1 (as far as I understand).

best,
Tim

On 04/29/2013 04:13 PM, Edward A. Berry wrote:
 herman.schreu...@sanofi.com wrote:
 I would process or expand the data to P1, also expand your pdb
 file to P1 and refine in P1 to see what happens. I would also run
 Phaser or some other molecular replacement program on the P1 data
 to see what comes out.
 
 
 process or expand? I don't understand how there can really be a
 choice here. If the data is expanded from higher symmetry, it will
 precisely have that higher symmetry, i.e. all reflections that were
 equivalent in the higher symmetry will be identical. Any
 information about asymmetry was lost when the data were merged in
 the higher symmetry space group. Won't refinement by minimizing a
 target function have exactly the same solution?
 
 Or not exactly, because the refinement program can introduce
 asymmetry by allowing different phases for the equivalent
 reflections. But where is the information to generate that
 asymmetry coming from?
 
 There is probably something I'm missing here, but i would
 definitely reprocess in the lower symmetry if the rotation angle
 was sufficient to give good completeness.
 
 eab
 

- -- 
- --
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A

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Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/

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nv3mtZH/Em2VlbDh9IFDvoU=
=F/Om
-END PGP SIGNATURE-

Re: [ccp4bb] refinement hanging--what am I missing?

[Phil Jeffrey]

Pat,

Try TLS - I usually don't invoke it at this type of resolution but in 
one case I saw it make a surprisingly significant improvement.


I would also be tempted to put the structures through Arp/wArp and see 
if it lowers the R-free any more - rightly or wrongly I view this as the 
lowest reasonably achievable R-factor with isotropic modeling - and 
especially look at the maps after it has finished in case it shows up 
anything you had missed.


When I had P21 - P2x212x twinning the R-free held up in the mid-30's at 
2 Angstrom resolution so absent any indications in Truncate or Xtriage I 
wouldn't suggest that.


A final question is how much disordered structure is missing from your 
models ?  Could a partly ordered but unmodeled segment be driving up 
R-free ?


Cheers
Phil Jeffrey
Princeton

On 4/26/13 5:38 PM, Patrick Loll wrote:

Hi all,

Here is a problem that's been annoying me, and demanding levels of thought all 
out of proportion with the importance of the project:

I have two related crystal forms of the same small protein. In both cases, the 
data look quite decent, and extend beyond 2 A, but the refinement stalls with 
statistics that are just bad enough to make me deeply uncomfortable. However, 
the maps look pretty good, and there's no obvious path to push the refinement 
further. Xtriage doesn't raise any red flags, nor does running the data through 
the Yeates twinning server.

Xtal form 1: P22(1)2(1), a=29.0, b=57.4, c=67.4; 2 molecules/AU. Resolution of 
data ~ 1.9 Å. Refinement converges with R/Rfree = 0.24/0.27

Xtal form 2: P2(1)2(1)2(1), a=59.50, b=61.1, c=67.2; 4 molecules/AU. Resolution 
of data ~ 1.7 Å. Refinement converges w/ R/Rfree = 0.21/0.26

As you would expect, the packing is essentially the same in both crystal forms.

It's interesting to note (but is it relevant?) that the packing is quite 
dense--solvent content is only 25-30%.

This kind of stalling at high R values smells like a twin problem, but it's not 
clear to me what specific kind of twinning might explain this behavior.

Any thoughts about what I might be missing here?

Thanks,

Pat


---
Patrick J. Loll, Ph. D.
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

Re: [ccp4bb] refinement hanging--what am I missing?

[Patrick Loll]

Responding to a couple of questions from Ethan, Charlie, and Phil:

Ethan: Using the default bulk solvent modeling in Phenix; no selenium, but I'll 
double-check wavelengths as a sanity check for scattering factors (but several 
other native data sets from the same synchrotron trip refined beautifully, so I 
suspect there's no gross boo-boos of this nature...)

Charlie:  Solvent regions are pretty clean; I haven't tried any flipping (these 
are molecular replacement models, so it didn't occur to me...). I tried 
applying NCS in one case (the smaller cell) and it had no apparent effect on 
the refinement. The Fo-Fc map has no strong features crying out for 
interpretation. Just based on geometry and map appearance, I'd be inclined to 
say the refinement is done, were it not for the crappy R values.

Phil:  I used TLS for refinement in both xtal forms; it gives a small 
improvement (0.01-0.03) in Rfree in both cases, but nothing magic. I simply 
used one monomer/TLS group (these are ubiquitin variants, so the monomer itself 
is pretty much a little rock, without any internal domain motions). There are 
the usual complement of disordered side chains, but nothing unusual, and  98% 
of the main chain is accounted for. Haven't tried Arp/wArp yet...

Excellent thoughts, keep those cards and letters coming. I'm still chewing on 
the substantive comments from Dean and Adrian...

Thanks,

Pat

On 26 Apr 2013, at 6:17 PM, Carter, Charlie wrote:

 Hi Pat,
 
 Your stats aren't all that bad, but I share your discomfort. 
 
 Do the solvent regions retain any significant features? Have you tried 
 flipping those features? Have you applied NCS? What does the Fo - Fc map look 
 like?
 
 Charlie
 
 On Apr 26, 2013, at 5:38 PM, Patrick Loll wrote:
 
 Hi all,
 
 Here is a problem that's been annoying me, and demanding levels of thought 
 all out of proportion with the importance of the project:
 
 I have two related crystal forms of the same small protein. In both cases, 
 the data look quite decent, and extend beyond 2 A, but the refinement stalls 
 with statistics that are just bad enough to make me deeply uncomfortable. 
 However, the maps look pretty good, and there's no obvious path to push the 
 refinement further. Xtriage doesn't raise any red flags, nor does running 
 the data through the Yeates twinning server.
 
 Xtal form 1: P22(1)2(1), a=29.0, b=57.4, c=67.4; 2 molecules/AU. Resolution 
 of data ~ 1.9 Å. Refinement converges with R/Rfree = 0.24/0.27
 
 Xtal form 2: P2(1)2(1)2(1), a=59.50, b=61.1, c=67.2; 4 molecules/AU. 
 Resolution of data ~ 1.7 Å. Refinement converges w/ R/Rfree = 0.21/0.26
 
 As you would expect, the packing is essentially the same in both crystal 
 forms. 
 
 It's interesting to note (but is it relevant?) that the packing is quite 
 dense--solvent content is only 25-30%.
 
 This kind of stalling at high R values smells like a twin problem, but it's 
 not clear to me what specific kind of twinning might explain this behavior.
 
 Any thoughts about what I might be missing here?
 
 Thanks,
 
 Pat
 
 
 ---
 Patrick J. Loll, Ph. D.  
 Professor of Biochemistry  Molecular Biology
 Director, Biochemistry Graduate Program
 Drexel University College of Medicine
 Room 10-102 New College Building
 245 N. 15th St., Mailstop 497
 Philadelphia, PA  19102-1192  USA
 
 (215) 762-7706
 pat.l...@drexelmed.edu
 

Re: [ccp4bb] refinement hanging--what am I missing?

[Boaz Shaanan]

Hi Pat,

On top of Phil's suggestion for invoking TLS, what about the NCS? Is there 
anything peculiar about them (tNCS or the like)? If there isn't, are you 
imposing NCS during refinement? perhaps you should relax the NCS restraints or 
not impose any at all and see what happens then?

My 2p thoughts.

Boaz


Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Israel

E-mail: bshaa...@bgu.ac.il
Phone: 972-8-647-2220  Skype: boaz.shaanan
Fax:   972-8-647-2992 or 972-8-646-1710






From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Patrick Loll 
[pat.l...@drexel.edu]
Sent: Saturday, April 27, 2013 12:38 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] refinement hanging--what am I missing?

Hi all,

Here is a problem that's been annoying me, and demanding levels of thought all 
out of proportion with the importance of the project:

I have two related crystal forms of the same small protein. In both cases, the 
data look quite decent, and extend beyond 2 A, but the refinement stalls with 
statistics that are just bad enough to make me deeply uncomfortable. However, 
the maps look pretty good, and there's no obvious path to push the refinement 
further. Xtriage doesn't raise any red flags, nor does running the data through 
the Yeates twinning server.

Xtal form 1: P22(1)2(1), a=29.0, b=57.4, c=67.4; 2 molecules/AU. Resolution of 
data ~ 1.9 Å. Refinement converges with R/Rfree = 0.24/0.27

Xtal form 2: P2(1)2(1)2(1), a=59.50, b=61.1, c=67.2; 4 molecules/AU. Resolution 
of data ~ 1.7 Å. Refinement converges w/ R/Rfree = 0.21/0.26

As you would expect, the packing is essentially the same in both crystal forms.

It's interesting to note (but is it relevant?) that the packing is quite 
dense--solvent content is only 25-30%.

This kind of stalling at high R values smells like a twin problem, but it's not 
clear to me what specific kind of twinning might explain this behavior.

Any thoughts about what I might be missing here?

Thanks,

Pat


---
Patrick J. Loll, Ph. D.
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

Re: [ccp4bb] refinement hanging--what am I missing?

[Robbie Joosten]

Hi Patrick,

Did you try using a different refinement program (e.g. Refmac)? Which type
of NCS restraints did you use, global or local (torsion- or distance-based)?
Have you tried optimizing your restraint weights? Have you tried running a
huge number of refinement cycles? You can also try running PDB_REDO (plug
plug) which will try a number of things to improve your model.

Cheers,
Robbie 

 -Original Message-
 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
 Patrick Loll
 Sent: Saturday, April 27, 2013 00:32
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] refinement hanging--what am I missing?
 
 Responding to a couple of questions from Ethan, Charlie, and Phil:
 
 Ethan: Using the default bulk solvent modeling in Phenix; no selenium, but
I'll
 double-check wavelengths as a sanity check for scattering factors (but
 several other native data sets from the same synchrotron trip refined
 beautifully, so I suspect there's no gross boo-boos of this nature...)
 
 Charlie:  Solvent regions are pretty clean; I haven't tried any flipping
(these
 are molecular replacement models, so it didn't occur to me...). I tried
 applying NCS in one case (the smaller cell) and it had no apparent effect
on
 the refinement. The Fo-Fc map has no strong features crying out for
 interpretation. Just based on geometry and map appearance, I'd be inclined
 to say the refinement is done, were it not for the crappy R values.
 
 Phil:  I used TLS for refinement in both xtal forms; it gives a small
 improvement (0.01-0.03) in Rfree in both cases, but nothing magic. I
simply
 used one monomer/TLS group (these are ubiquitin variants, so the monomer
 itself is pretty much a little rock, without any internal domain motions).
There
 are the usual complement of disordered side chains, but nothing unusual,
 and  98% of the main chain is accounted for. Haven't tried Arp/wArp
yet...
 
 Excellent thoughts, keep those cards and letters coming. I'm still chewing
on
 the substantive comments from Dean and Adrian...
 
 Thanks,
 
 Pat
 
 On 26 Apr 2013, at 6:17 PM, Carter, Charlie wrote:
 
  Hi Pat,
 
  Your stats aren't all that bad, but I share your discomfort.
 
  Do the solvent regions retain any significant features? Have you tried
 flipping those features? Have you applied NCS? What does the Fo - Fc map
 look like?
 
  Charlie
 
  On Apr 26, 2013, at 5:38 PM, Patrick Loll wrote:
 
  Hi all,
 
  Here is a problem that's been annoying me, and demanding levels of
 thought all out of proportion with the importance of the project:
 
  I have two related crystal forms of the same small protein. In both
cases,
 the data look quite decent, and extend beyond 2 A, but the refinement
stalls
 with statistics that are just bad enough to make me deeply uncomfortable.
 However, the maps look pretty good, and there's no obvious path to push
 the refinement further. Xtriage doesn't raise any red flags, nor does
running
 the data through the Yeates twinning server.
 
  Xtal form 1: P22(1)2(1), a=29.0, b=57.4, c=67.4; 2 molecules/AU.
  Resolution of data ~ 1.9 Å. Refinement converges with R/Rfree =
  0.24/0.27
 
  Xtal form 2: P2(1)2(1)2(1), a=59.50, b=61.1, c=67.2; 4 molecules/AU.
  Resolution of data ~ 1.7 Å. Refinement converges w/ R/Rfree =
  0.21/0.26
 
  As you would expect, the packing is essentially the same in both
crystal
 forms.
 
  It's interesting to note (but is it relevant?) that the packing is
quite dense-
 -solvent content is only 25-30%.
 
  This kind of stalling at high R values smells like a twin problem, but
it's not
 clear to me what specific kind of twinning might explain this behavior.
 
  Any thoughts about what I might be missing here?
 
  Thanks,
 
  Pat
 
 
  -
  --
  Patrick J. Loll, Ph. D.
  Professor of Biochemistry  Molecular Biology Director, Biochemistry
  Graduate Program Drexel University College of Medicine Room 10-102
  New College Building
  245 N. 15th St., Mailstop 497
  Philadelphia, PA  19102-1192  USA
 
  (215) 762-7706
  pat.l...@drexelmed.edu
 

Re: [ccp4bb] Refinement with anomalous signal

[Kavyashree Manjunath]

Sir,

Thank you Sir. I tried this once at the end in order to
check the refinement statistics, R, Rfree and FOM showed
improvement. but I encountered one problem. One of the a
nomalous scatters which had double occupancies (which was
confirmed by anomalous peak search) after the refinement
of occupancy using SAD data directly it turned out that
the sum of the refined occupancies of this atom was more
than 1. Why is it so? What might have gone wrong here?

Regards
Kavya

 Hi Kavya,

 In my experience, if the SAD data are good, in addition to helping with
 anomalous scatterer occupancy refinement the R factors can be
 significantly
 improved as well. I would give it a try. As far as the other options in
 refmac such as inclusion of H-L coefficients or phase, FOM, I believe that
 direct refinement against the SAD data is preferred.

 Philip


 On Fri, Apr 19, 2013 at 7:43 AM, Kavyashree Manjunath 
 ka...@ssl.serc.iisc.in wrote:

 Dear users,

 The native structure for a protein is available and there is a
 ligand bound data. The crystallisation condition has anomalous
 scattering metal ions (Cd). Both the data are scaled by separating
 anomalous pairs. So while refining a ligand bound data with a
 solution obtained using Molecular replacement, is it recommended
 to refine using SAD data directly in refmac so that the anomalous
 atoms can be occupancy refined?

 Thanking you
 Regards
 Kavya


 --
 This message has been scanned for viruses and
 dangerous content by MailScanner, and is
 believed to be clean.




 --
 Philip D. Kiser, Pharm.D., Ph.D.
 Department of Pharmacology
 Case Western Reserve University
 10900 Euclid Ave. Wood Building Room 317
 Cleveland, OH 44106
 (216) 368-8794

 --
 This message has been scanned for viruses and
 dangerous content by MailScanner, and is
 believed to be clean.





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Re: [ccp4bb] Refinement with anomalous signal

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Kavya,

one reason could be an incorrect script for running refmac, or a bug
in refmac, or, if the distance allows, that the two peaks really are
two different fully occupied atoms with a lesser anomalous signal than
expected.

Best,
Tim

On 04/19/2013 02:02 PM, Kavyashree Manjunath wrote:
 Sir,
 
 Thank you Sir. I tried this once at the end in order to check the
 refinement statistics, R, Rfree and FOM showed improvement. but I
 encountered one problem. One of the a nomalous scatters which had
 double occupancies (which was confirmed by anomalous peak search)
 after the refinement of occupancy using SAD data directly it
 turned out that the sum of the refined occupancies of this atom was
 more than 1. Why is it so? What might have gone wrong here?
 
 Regards Kavya
 
 Hi Kavya,
 
 In my experience, if the SAD data are good, in addition to
 helping with anomalous scatterer occupancy refinement the R
 factors can be significantly improved as well. I would give it a
 try. As far as the other options in refmac such as inclusion of
 H-L coefficients or phase, FOM, I believe that direct refinement
 against the SAD data is preferred.
 
 Philip
 
 
 On Fri, Apr 19, 2013 at 7:43 AM, Kavyashree Manjunath  
 ka...@ssl.serc.iisc.in wrote:
 
 Dear users,
 
 The native structure for a protein is available and there is a 
 ligand bound data. The crystallisation condition has anomalous 
 scattering metal ions (Cd). Both the data are scaled by
 separating anomalous pairs. So while refining a ligand bound
 data with a solution obtained using Molecular replacement, is
 it recommended to refine using SAD data directly in refmac so
 that the anomalous atoms can be occupancy refined?
 
 Thanking you Regards Kavya
 
 
 -- This message has been scanned for viruses and dangerous
 content by MailScanner, and is believed to be clean.
 
 
 
 
 -- Philip D. Kiser, Pharm.D., Ph.D. Department of Pharmacology 
 Case Western Reserve University 10900 Euclid Ave. Wood Building
 Room 317 Cleveland, OH 44106 (216) 368-8794
 
 -- This message has been scanned for viruses and dangerous
 content by MailScanner, and is believed to be clean.
 
 
 
 
 

- -- 
- --
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A

-BEGIN PGP SIGNATURE-
Version: GnuPG v1.4.12 (GNU/Linux)
Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/

iD8DBQFRcTNHUxlJ7aRr7hoRAh2NAKCs2RCLwJfGjRUPwJrKIAhb2/ucBACg17+/
JvNU5K2lQ1jHnu/aHQ9TK94=
=1zQO
-END PGP SIGNATURE-

Re: [ccp4bb] Refinement with anomalous signal

[Kavyashree Manjunath]

Dear Sir,

Thank you. I run refmac using GUI so most
Probably there are two different atoms.

Regards
Kavya

 -BEGIN PGP SIGNED MESSAGE-
 Hash: SHA1

 Dear Kavya,

 one reason could be an incorrect script for running refmac, or a bug
 in refmac, or, if the distance allows, that the two peaks really are
 two different fully occupied atoms with a lesser anomalous signal than
 expected.

 Best,
 Tim

 On 04/19/2013 02:02 PM, Kavyashree Manjunath wrote:
 Sir,

 Thank you Sir. I tried this once at the end in order to check the
 refinement statistics, R, Rfree and FOM showed improvement. but I
 encountered one problem. One of the a nomalous scatters which had
 double occupancies (which was confirmed by anomalous peak search)
 after the refinement of occupancy using SAD data directly it
 turned out that the sum of the refined occupancies of this atom was
 more than 1. Why is it so? What might have gone wrong here?

 Regards Kavya

 Hi Kavya,

 In my experience, if the SAD data are good, in addition to
 helping with anomalous scatterer occupancy refinement the R
 factors can be significantly improved as well. I would give it a
 try. As far as the other options in refmac such as inclusion of
 H-L coefficients or phase, FOM, I believe that direct refinement
 against the SAD data is preferred.

 Philip


 On Fri, Apr 19, 2013 at 7:43 AM, Kavyashree Manjunath 
 ka...@ssl.serc.iisc.in wrote:

 Dear users,

 The native structure for a protein is available and there is a
 ligand bound data. The crystallisation condition has anomalous
 scattering metal ions (Cd). Both the data are scaled by
 separating anomalous pairs. So while refining a ligand bound
 data with a solution obtained using Molecular replacement, is
 it recommended to refine using SAD data directly in refmac so
 that the anomalous atoms can be occupancy refined?

 Thanking you Regards Kavya


 -- This message has been scanned for viruses and dangerous
 content by MailScanner, and is believed to be clean.




 -- Philip D. Kiser, Pharm.D., Ph.D. Department of Pharmacology
 Case Western Reserve University 10900 Euclid Ave. Wood Building
 Room 317 Cleveland, OH 44106 (216) 368-8794

 -- This message has been scanned for viruses and dangerous
 content by MailScanner, and is believed to be clean.






 - --
 - --
 Dr Tim Gruene
 Institut fuer anorganische Chemie
 Tammannstr. 4
 D-37077 Goettingen

 GPG Key ID = A46BEE1A

 -BEGIN PGP SIGNATURE-
 Version: GnuPG v1.4.12 (GNU/Linux)
 Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/

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Re: [ccp4bb] refinement protein structure

[Savvas Savvides]

Dear Tom
in addition to the very valuable input you have received already, I would like 
to point out that based on the fact that the I/sigma_I of your data is above 
3 in the highest resolution shell, you will likely be able to push the 
resolution limits of your analysis to go beyond 1.4 angs resolution.  I would 
definitely enoucrage you to do so even if Rmeas values end up being higher than 
what common practice and rules of thumb may prescribe. 
Recent work by Karplus and Diederichs (Science 336, 1030 (2012)) and an 
accompanying editorial by Phil Evans (Science 336, 986 (2012)) will provide the 
necessary food for thought to proceed.

Very best wishes
Savvas


Savvas Savvides
Unit for Structural Biology, L-ProBE
Ghent University
K.L. Ledeganckstraat 35, 9000 Ghent, Belgium
Tel/SMS/texting +32  (0)472 928 519
Skype: savvas.savvides_skype
http://www.LProBE.ugent.be/xray.html



On 27 Mar 2013, at 17:22, Tom Van den Bergh 
tom.vandenbe...@student.kuleuven.be wrote:

 Dear members of ccp4bb,
 
 I need some help with the refinement of my structure of a variant of mRFP 
 (monomer red fluorescent protein, sequence in attachment). I have done 
 molecular replacement with phaser with model 2VAD of protein database. Then i 
 have done some model building phenix.autobuild. (2 pdb's (overall...), freeR 
 flags and log file attached) When i refine with phenix.refine my structure i 
 get a R-value of 0,42 which is still way too high. (redfluorescent 
 protein.pdb, .mtz and logfile attached) When i look at the structure in coot 
 i find many unmodelled blobs and many outliers in density analysis and 
 rotamer analysis. The problem is that there are so many problems with my 
 structure, that i dont know where to begin. Could you try some refinement for 
 me, because this is first structure that i need to solve as a student and i 
 dont have too many experience with it.
 
 Greetings,
 
 Tom
 
 overall_best.pdbexptl_fobs_phases_freeR_flags.mtzoverall_best_placed.pdbredfluorescentprotein_refine_10.pdbredfluorescentprotein_refine_10.mtzmRFPsequentiezondertag.fastaredfluorescentprotein_refine_10.logAutoBuild_run_6_1.log

Re: [ccp4bb] refinement protein structure

[Petr Leiman]

Dear Tom,

As far as I know you are the first person who sent his/her data that are 
supposed to be _private_ to 1000+ CCP4BB subscribers.

The person who suggested you send this type of message to CCP4BB board played a 
very cruel practical joke on you.

To CCP4BB support staff: Is it possible to block emails with 1+MB attachments?

Thank you,

Petr


Prof. Petr Leiman
EPFL
BSP-415
CH-1015 Lausanne
Switzerland

On Mar 27, 2013, at 5:22 PM, Tom Van den Bergh wrote:

Dear members of ccp4bb,

I need some help with the refinement of my structure of a variant of mRFP 
(monomer red fluorescent protein, sequence in attachment). I have done 
molecular replacement with phaser with model 2VAD of protein database. Then i 
have done some model building phenix.autobuild. (2 pdb's (overall...), freeR 
flags and log file attached) When i refine with phenix.refine my structure i 
get a R-value of 0,42 which is still way too high. (redfluorescent protein.pdb, 
.mtz and logfile attached) When i look at the structure in coot i find many 
unmodelled blobs and many outliers in density analysis and rotamer analysis. 
The problem is that there are so many problems with my structure, that i dont 
know where to begin. Could you try some refinement for me, because this is 
first structure that i need to solve as a student and i dont have too many 
experience with it.

Greetings,

Tom

overall_best.pdbexptl_fobs_phases_freeR_flags.mtzoverall_best_placed.pdbredfluorescentprotein_refine_10.pdbredfluorescentprotein_refine_10.mtzmRFPsequentiezondertag.fastaredfluorescentprotein_refine_10.logAutoBuild_run_6_1.log

Re: [ccp4bb] refinement protein structure

[Antony Oliver]

Dear Tom,

Q1: Are you really supposed to posting this information here?
Q2: Is there really NOT anyone you can ask in your own department to help you 
with this?

Some initial hints/ideas/suggestions though - although I actually assume this 
is meant to be a learning exercise for you …?

1) Have you got the correct spacegroup?
2) Have you got enough molecules in the asymmetric unit?
3) Do your placed molecules pack together to form a sensible crystal lattice?

Tony.

On 27 Mar 2013, at 16:22, Tom Van den Bergh 
tom.vandenbe...@student.kuleuven.bemailto:tom.vandenbe...@student.kuleuven.be
 wrote:

Dear members of ccp4bb,

I need some help with the refinement of my structure of a variant of mRFP 
(monomer red fluorescent protein, sequence in attachment). I have done 
molecular replacement with phaser with model 2VAD of protein database. Then i 
have done some model building phenix.autobuild. (2 pdb's (overall...), freeR 
flags and log file attached) When i refine with phenix.refine my structure i 
get a R-value of 0,42 which is still way too high. (redfluorescent protein.pdb, 
.mtz and logfile attached) When i look at the structure in coot i find many 
unmodelled blobs and many outliers in density analysis and rotamer analysis. 
The problem is that there are so many problems with my structure, that i dont 
know where to begin. Could you try some refinement for me, because this is 
first structure that i need to solve as a student and i dont have too many 
experience with it.

Greetings,

Tom

overall_best.pdbexptl_fobs_phases_freeR_flags.mtzoverall_best_placed.pdbredfluorescentprotein_refine_10.pdbredfluorescentprotein_refine_10.mtzmRFPsequentiezondertag.fastaredfluorescentprotein_refine_10.logAutoBuild_run_6_1.log

Re: [ccp4bb] refinement protein structure

[Eugene Osipov]

Dear Tom,
I think that only way for you is to make this structure yourself because it
is only way to learn something (at first you must ask in your lab).
Anyway, as student I can make some brief suggestions and guides for your
refinement
1) Find as much info about your protein as possible, especially look at
previous structure (read 2VAD related structure as first step)
2) Before refinement you must find conservative domains inside your protein
or in other words red fluorescent protein motifs - these residues will be
your starting point
3) As this motif is conserved and probably MR will place them in correct
orientation you must move forth and appropriately mutate incorrect
residues, if their CA fits good. If your density becomes bad you will
remove incorrect residues from this point, later by FOFC map you will place
the rest of them in correct orientation. Now move back from the conserved
motif in the same manner.
4) After this run 8-10 rounds of refinement, generate map. Repeat step 3
Hope this will help you somehow

Important note for you: do not show your data before publication of pdb.

2013/3/27 Tom Van den Bergh tom.vandenbe...@student.kuleuven.be

  Dear members of ccp4bb,

 I need some help with the refinement of my structure of a variant of mRFP
 (monomer red fluorescent protein, sequence in attachment). I have done
 molecular replacement with phaser with model 2VAD of protein database. Then
 i have done some model building phenix.autobuild. (2 pdb's (overall...),
 freeR flags and log file attached) When i refine with phenix.refine my
 structure i get a R-value of 0,42 which is still way too high.
 (redfluorescent protein.pdb, .mtz and logfile attached) When i look at the
 structure in coot i find many unmodelled blobs and many outliers in density
 analysis and rotamer analysis. The problem is that there are so many
 problems with my structure, that i dont know where to begin. Could you try
 some refinement for me, because this is first structure that i need to
 solve as a student and i dont have too many experience with it.

 Greetings,

 Tom




-- 
Eugene Osipov
Junior Research Scientist
Laboratory of Enzyme Engineering
A.N. Bach Institute of Biochemistry
Russian Academy of Sciences
Leninsky pr. 33, 119071 Moscow, Russia
e-mail: e.m.osi...@gmail.com

Re: [ccp4bb] refinement protein structure

[Phil Jeffrey]

That's quite brave - shipping your entire structure to people that could 
be actual competitors.  But it was fun to play at 1.4 Angstrom over lunch.


Practical points:

* not everyone loves 12Mb of attachments in one email in their inbox, so 
if you do this again please put the files on a webserver and point us there


Structural points:

* the map looks pretty good, but I think the sequence is misassigned in 
some regions (e.g. A118-A122 etc).  Automation is a good tool but a poor 
master, and extreme caution is required before taking the results too 
literally.  Usually you'd expect a 1.4 Angstrom to be easy to autobuild 
but I recently had a sequence misassignment at just that resolution. 
That map was trivial to interpret with the correct sequence however - 
one of the joys of working with Arp/wArp at 1.4 Angstrom.


* the large number of positive difference density blobs and water 
molecules clustered in what otherwise would be the solvent void strongly 
suggest that there's a second molecule present.



If I take redfluorescentprotein_refine_10.pdb (waters removed) and 
exptl_fobs_phases_freeR_flags.mtz and ask Phaser to look for two 
molecules, it finds them quite successfully.  (for the record an LLG of 
15111 using nominal sequence identity of 90%).  I will send this to you 
off-list.  Please note that Phaser is using a different origin for this 
molecular replacement solution so the coordinates and your previous map 
do not overlap.


This rather nicely explains why your structure had an R-factor in the 
40's despite being a half-way decent model.  The new MR solution has an 
R-free in the 30's in the phenix.refine job I'm running right now.



Going forward I suggest you utilize the Arp/wArp program to autobuild 
your structure for you, starting from the molecular replacement solution 
(or, perhaps with it stripped to ALA).  While you could use Autobuild, 
this is the CCP4 list and so you should use CCP4 programs.


Phil Jeffrey
Princeton


On 3/27/13 12:22 PM, Tom Van den Bergh wrote:

Dear members of ccp4bb,

I need some help with the refinement of my structure of a variant of
mRFP (monomer red fluorescent protein, sequence in attachment). I have
done molecular replacement with phaser with model 2VAD of protein
database. Then i have done some model building phenix.autobuild. (2
pdb's (overall...), freeR flags and log file attached) When i refine
with phenix.refine my structure i get a R-value of 0,42 which is still
way too high. (redfluorescent protein.pdb, .mtz and logfile attached)
When i look at the structure in coot i find many unmodelled blobs and
many outliers in density analysis and rotamer analysis. The problem is
that there are so many problems with my structure, that i dont know
where to begin. Could you try some refinement for me, because this is
first structure that i need to solve as a student and i dont have too
many experience with it.

Greetings,

Tom

Re: [ccp4bb] refinement protein structure

[Petr Leiman]

Since this is now public domain knowledge and if this gets ever published, Phil 
has my vote to be the first author!

Petr


On Mar 27, 2013, at 6:09 PM, Phil Jeffrey wrote:

 That's quite brave - shipping your entire structure to people that could be 
 actual competitors.  But it was fun to play at 1.4 Angstrom over lunch.
 
 Practical points:
 
 * not everyone loves 12Mb of attachments in one email in their inbox, so if 
 you do this again please put the files on a webserver and point us there
 
 Structural points:
 
 * the map looks pretty good, but I think the sequence is misassigned in some 
 regions (e.g. A118-A122 etc).  Automation is a good tool but a poor master, 
 and extreme caution is required before taking the results too literally.  
 Usually you'd expect a 1.4 Angstrom to be easy to autobuild but I recently 
 had a sequence misassignment at just that resolution. That map was trivial to 
 interpret with the correct sequence however - one of the joys of working with 
 Arp/wArp at 1.4 Angstrom.
 
 * the large number of positive difference density blobs and water molecules 
 clustered in what otherwise would be the solvent void strongly suggest that 
 there's a second molecule present.
 
 
 If I take redfluorescentprotein_refine_10.pdb (waters removed) and 
 exptl_fobs_phases_freeR_flags.mtz and ask Phaser to look for two molecules, 
 it finds them quite successfully.  (for the record an LLG of 15111 using 
 nominal sequence identity of 90%).  I will send this to you off-list.  Please 
 note that Phaser is using a different origin for this molecular replacement 
 solution so the coordinates and your previous map do not overlap.
 
 This rather nicely explains why your structure had an R-factor in the 40's 
 despite being a half-way decent model.  The new MR solution has an R-free in 
 the 30's in the phenix.refine job I'm running right now.
 
 
 Going forward I suggest you utilize the Arp/wArp program to autobuild your 
 structure for you, starting from the molecular replacement solution (or, 
 perhaps with it stripped to ALA).  While you could use Autobuild, this is the 
 CCP4 list and so you should use CCP4 programs.
 
 Phil Jeffrey
 Princeton
 
 
 On 3/27/13 12:22 PM, Tom Van den Bergh wrote:
 Dear members of ccp4bb,
 
 I need some help with the refinement of my structure of a variant of
 mRFP (monomer red fluorescent protein, sequence in attachment). I have
 done molecular replacement with phaser with model 2VAD of protein
 database. Then i have done some model building phenix.autobuild. (2
 pdb's (overall...), freeR flags and log file attached) When i refine
 with phenix.refine my structure i get a R-value of 0,42 which is still
 way too high. (redfluorescent protein.pdb, .mtz and logfile attached)
 When i look at the structure in coot i find many unmodelled blobs and
 many outliers in density analysis and rotamer analysis. The problem is
 that there are so many problems with my structure, that i dont know
 where to begin. Could you try some refinement for me, because this is
 first structure that i need to solve as a student and i dont have too
 many experience with it.
 
 Greetings,
 
 Tom
 

Re: [ccp4bb] refinement protein structure

[Antony Oliver]

At the risk of being somewhat cheeky - perhaps I could claim second author?
I too have successfully solved the structure - and I totally concur with Phil.
Placing a second molecule in the asymmetric unit, essentially resolves the 
perceived R-factor problem.

A good thorough manual inspection and rebuilding is *ALWAYS* good practice for 
newcomers to the field.

Tony.


On 27 Mar 2013, at 17:14, Petr Leiman petr.lei...@epfl.ch
 wrote:

 Since this is now public domain knowledge and if this gets ever published, 
 Phil has my vote to be the first author!
 
 Petr
 
 
 On Mar 27, 2013, at 6:09 PM, Phil Jeffrey wrote:
 
 That's quite brave - shipping your entire structure to people that could be 
 actual competitors.  But it was fun to play at 1.4 Angstrom over lunch.
 
 Practical points:
 
 * not everyone loves 12Mb of attachments in one email in their inbox, so if 
 you do this again please put the files on a webserver and point us there
 
 Structural points:
 
 * the map looks pretty good, but I think the sequence is misassigned in some 
 regions (e.g. A118-A122 etc).  Automation is a good tool but a poor master, 
 and extreme caution is required before taking the results too literally.  
 Usually you'd expect a 1.4 Angstrom to be easy to autobuild but I recently 
 had a sequence misassignment at just that resolution. That map was trivial 
 to interpret with the correct sequence however - one of the joys of working 
 with Arp/wArp at 1.4 Angstrom.
 
 * the large number of positive difference density blobs and water molecules 
 clustered in what otherwise would be the solvent void strongly suggest that 
 there's a second molecule present.
 
 
 If I take redfluorescentprotein_refine_10.pdb (waters removed) and 
 exptl_fobs_phases_freeR_flags.mtz and ask Phaser to look for two molecules, 
 it finds them quite successfully.  (for the record an LLG of 15111 using 
 nominal sequence identity of 90%).  I will send this to you off-list.  
 Please note that Phaser is using a different origin for this molecular 
 replacement solution so the coordinates and your previous map do not overlap.
 
 This rather nicely explains why your structure had an R-factor in the 40's 
 despite being a half-way decent model.  The new MR solution has an R-free in 
 the 30's in the phenix.refine job I'm running right now.
 
 
 Going forward I suggest you utilize the Arp/wArp program to autobuild your 
 structure for you, starting from the molecular replacement solution (or, 
 perhaps with it stripped to ALA).  While you could use Autobuild, this is 
 the CCP4 list and so you should use CCP4 programs.
 
 Phil Jeffrey
 Princeton
 
 
 On 3/27/13 12:22 PM, Tom Van den Bergh wrote:
 Dear members of ccp4bb,
 
 I need some help with the refinement of my structure of a variant of
 mRFP (monomer red fluorescent protein, sequence in attachment). I have
 done molecular replacement with phaser with model 2VAD of protein
 database. Then i have done some model building phenix.autobuild. (2
 pdb's (overall...), freeR flags and log file attached) When i refine
 with phenix.refine my structure i get a R-value of 0,42 which is still
 way too high. (redfluorescent protein.pdb, .mtz and logfile attached)
 When i look at the structure in coot i find many unmodelled blobs and
 many outliers in density analysis and rotamer analysis. The problem is
 that there are so many problems with my structure, that i dont know
 where to begin. Could you try some refinement for me, because this is
 first structure that i need to solve as a student and i dont have too
 many experience with it.
 
 Greetings,
 
 Tom
 

Re: [ccp4bb] refinement protein structure

[Steiner, Roberto]

we should all chip in a water molecule or two and the second author becomes 
the CCP4 community….

On 27 Mar 2013, at 17:22, Antony Oliver antony.oli...@sussex.ac.uk
 wrote:

 At the risk of being somewhat cheeky - perhaps I could claim second author?
 I too have successfully solved the structure - and I totally concur with Phil.
 Placing a second molecule in the asymmetric unit, essentially resolves the 
 perceived R-factor problem.
 
 A good thorough manual inspection and rebuilding is *ALWAYS* good practice 
 for newcomers to the field.
 
 Tony.
 
 
 On 27 Mar 2013, at 17:14, Petr Leiman petr.lei...@epfl.ch
 wrote:
 
 Since this is now public domain knowledge and if this gets ever published, 
 Phil has my vote to be the first author!
 
 Petr
 
 
 On Mar 27, 2013, at 6:09 PM, Phil Jeffrey wrote:
 
 That's quite brave - shipping your entire structure to people that could be 
 actual competitors.  But it was fun to play at 1.4 Angstrom over lunch.
 
 Practical points:
 
 * not everyone loves 12Mb of attachments in one email in their inbox, so if 
 you do this again please put the files on a webserver and point us there
 
 Structural points:
 
 * the map looks pretty good, but I think the sequence is misassigned in 
 some regions (e.g. A118-A122 etc).  Automation is a good tool but a poor 
 master, and extreme caution is required before taking the results too 
 literally.  Usually you'd expect a 1.4 Angstrom to be easy to autobuild but 
 I recently had a sequence misassignment at just that resolution. That map 
 was trivial to interpret with the correct sequence however - one of the 
 joys of working with Arp/wArp at 1.4 Angstrom.
 
 * the large number of positive difference density blobs and water molecules 
 clustered in what otherwise would be the solvent void strongly suggest that 
 there's a second molecule present.
 
 
 If I take redfluorescentprotein_refine_10.pdb (waters removed) and 
 exptl_fobs_phases_freeR_flags.mtz and ask Phaser to look for two molecules, 
 it finds them quite successfully.  (for the record an LLG of 15111 using 
 nominal sequence identity of 90%).  I will send this to you off-list.  
 Please note that Phaser is using a different origin for this molecular 
 replacement solution so the coordinates and your previous map do not 
 overlap.
 
 This rather nicely explains why your structure had an R-factor in the 40's 
 despite being a half-way decent model.  The new MR solution has an R-free 
 in the 30's in the phenix.refine job I'm running right now.
 
 
 Going forward I suggest you utilize the Arp/wArp program to autobuild your 
 structure for you, starting from the molecular replacement solution (or, 
 perhaps with it stripped to ALA).  While you could use Autobuild, this is 
 the CCP4 list and so you should use CCP4 programs.
 
 Phil Jeffrey
 Princeton
 
 
 On 3/27/13 12:22 PM, Tom Van den Bergh wrote:
 Dear members of ccp4bb,
 
 I need some help with the refinement of my structure of a variant of
 mRFP (monomer red fluorescent protein, sequence in attachment). I have
 done molecular replacement with phaser with model 2VAD of protein
 database. Then i have done some model building phenix.autobuild. (2
 pdb's (overall...), freeR flags and log file attached) When i refine
 with phenix.refine my structure i get a R-value of 0,42 which is still
 way too high. (redfluorescent protein.pdb, .mtz and logfile attached)
 When i look at the structure in coot i find many unmodelled blobs and
 many outliers in density analysis and rotamer analysis. The problem is
 that there are so many problems with my structure, that i dont know
 where to begin. Could you try some refinement for me, because this is
 first structure that i need to solve as a student and i dont have too
 many experience with it.
 
 Greetings,
 
 Tom
 
 

Roberto A. Steiner
Group Leader
Randall Division of Cell and Molecular Biophysics
King's College London
roberto.stei...@kcl.ac.uk

Room 3.10A
New Hunt's House
Guy's Campus
SE1 1UL
London

Phone 0044 20 78488216
Fax0044 20 78486435

Re: [ccp4bb] refinement protein structure

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear so-far-posters,

I do not know Tom Van den Bergh, nor do I know his background, nor the
history of the data, nor the reasons why he may have sent it to this
list (although I think he did it to ask for help), but I find these
answers irritatingly disrespectful and nasty.

No regards to the ones addressed,
Tim

On 03/27/2013 06:43 PM, Bosch, Juergen wrote:
 Sorry it has been accepted already, see attachment, it will soon be
 online see the date for that.
 
 Jürgen
 
 .. Jürgen Bosch Johns Hopkins University 
 Bloomberg School of Public Health Department of Biochemistry 
 Molecular Biology Johns Hopkins Malaria Research Institute 615
 North Wolfe Street, W8708 Baltimore, MD 21205 Office:
 +1-410-614-4742 Lab:  +1-410-614-4894 Fax:
 +1-410-955-2926 http://lupo.jhsph.edu
 
 [cid:C2A945C2-7702-4EAE-B6E5-2825754D728C@sph.ad.jhsph.edu] On Mar
 27, 2013, at 1:30 PM, Steiner, Roberto wrote:
 
 we should all chip in a water molecule or two and the second author
 becomes the CCP4 community….
 
 On 27 Mar 2013, at 17:22, Antony Oliver
 antony.oli...@sussex.ac.ukmailto:antony.oli...@sussex.ac.uk 
 wrote:
 
 At the risk of being somewhat cheeky - perhaps I could claim second
 author? I too have successfully solved the structure - and I
 totally concur with Phil. Placing a second molecule in the
 asymmetric unit, essentially resolves the perceived R-factor
 problem.
 
 A good thorough manual inspection and rebuilding is *ALWAYS* good
 practice for newcomers to the field.
 
 Tony.
 
 
 On 27 Mar 2013, at 17:14, Petr Leiman
 petr.lei...@epfl.chmailto:petr.lei...@epfl.ch wrote:
 
 Since this is now public domain knowledge and if this gets ever
 published, Phil has my vote to be the first author!
 
 Petr
 
 
 On Mar 27, 2013, at 6:09 PM, Phil Jeffrey wrote:
 
 That's quite brave - shipping your entire structure to people that
 could be actual competitors.  But it was fun to play at 1.4
 Angstrom over lunch.
 
 Practical points:
 
 * not everyone loves 12Mb of attachments in one email in their
 inbox, so if you do this again please put the files on a webserver
 and point us there
 
 Structural points:
 
 * the map looks pretty good, but I think the sequence is
 misassigned in some regions (e.g. A118-A122 etc).  Automation is a
 good tool but a poor master, and extreme caution is required before
 taking the results too literally.  Usually you'd expect a 1.4
 Angstrom to be easy to autobuild but I recently had a sequence
 misassignment at just that resolution. That map was trivial to
 interpret with the correct sequence however - one of the joys of
 working with Arp/wArp at 1.4 Angstrom.
 
 * the large number of positive difference density blobs and water
 molecules clustered in what otherwise would be the solvent void
 strongly suggest that there's a second molecule present.
 
 
 If I take redfluorescentprotein_refine_10.pdb (waters removed) and
 exptl_fobs_phases_freeR_flags.mtz and ask Phaser to look for two
 molecules, it finds them quite successfully.  (for the record an
 LLG of 15111 using nominal sequence identity of 90%).  I will send
 this to you off-list.  Please note that Phaser is using a different
 origin for this molecular replacement solution so the coordinates
 and your previous map do not overlap.
 
 This rather nicely explains why your structure had an R-factor in
 the 40's despite being a half-way decent model.  The new MR
 solution has an R-free in the 30's in the phenix.refine job I'm
 running right now.
 
 
 Going forward I suggest you utilize the Arp/wArp program to
 autobuild your structure for you, starting from the molecular
 replacement solution (or, perhaps with it stripped to ALA).  While
 you could use Autobuild, this is the CCP4 list and so you should
 use CCP4 programs.
 
 Phil Jeffrey Princeton
 
 
 On 3/27/13 12:22 PM, Tom Van den Bergh wrote: Dear members of
 ccp4bb,
 
 I need some help with the refinement of my structure of a variant
 of mRFP (monomer red fluorescent protein, sequence in attachment).
 I have done molecular replacement with phaser with model 2VAD of
 protein database. Then i have done some model building
 phenix.autobuild. (2 pdb's (overall...), freeR flags and log file
 attached) When i refine with phenix.refine my structure i get a
 R-value of 0,42 which is still way too high. (redfluorescent
 protein.pdb, .mtz and logfile attached) When i look at the
 structure in coot i find many unmodelled blobs and many outliers in
 density analysis and rotamer analysis. The problem is that there
 are so many problems with my structure, that i dont know where to
 begin. Could you try some refinement for me, because this is first
 structure that i need to solve as a student and i dont have too 
 many experience with it.
 
 Greetings,
 
 Tom
 
 
 
 Roberto A. Steiner Group Leader Randall Division of Cell and
 Molecular Biophysics King's College London 
 roberto.stei...@kcl.ac.ukmailto:roberto.stei...@kcl.ac.uk
 
 

Re: [ccp4bb] refinement protein structure

[Roger Rowlett]

Tom,

Welcome to the protein crystallography community. I hope you take all 
the teasing in good humor. And a few protocol lessons about sharing 
data, etc. By now you have discovered that structure factor files and a 
possible MR model are like catnip to structural biologists. I learned 
this when I solved my first structure while on a sabbatical leave. I ask 
one question about a tricky (to me) MR solution, share the structure 
factors with ONE postdoc, and the next thing I know the whole building 
is solving my structure. (Cover ears and go La La La La La.) You have 
taken this to a whole new level! :)


To echo the advice of many others, nothing beats actually looking at the 
solution with a critical eye:


1. is there sensible electron density around the model? (You may want
   to modify the model to poly-Ala or truncate to the nearest similar
   residue--you can do this using CCP4 programs.) If sequence identity
   is really poor a poly-Ala search model may be better. Once you get a
   preliminary solution, I've used Parrot and Buccaneer successfully to
   autobuild most of the structure from a relatively poor starting
   model. But there is nothing wrong with manual rebuilding from a good
   starting point with high homology.
2. Does the solution pack well, with definable solvent channels and
   reasonable protein-protein contacts? (Use symexp in Pymol or turn on
   symmetry molecules in Coot) Sometimes it is easy to discover a
   missing or extra molecule in the ASU this way. That's what
   happened with my first structure solution--I thought I had 4 chains
   in the ASU and it was really 6, which was quite obvious from the
   packing of my partial (as it turned out) MR solution. An extra two
   chains fit perfectly in the hole in the packing view.
3. Don't forget to do a quick Matthews coefficient calculation before
   MR to get some idea of how many molecules are in the ASU. It's not
   perfect, especially for high copy numbers, but it's a start. It's
   normally easy to tell 1 from 2 molecules in the ASU. Not so much 6
   vs. 8.
4. It's not unusual for raw MR solutions to start at R40%. But if you
   are on the right track, the initial refinement in Refmac should
   drive that down to 30% or lower pretty quickly. If R doesn't drop
   rapidly, you likely have an issue with the MR solution. Be aware
   that in the latest versions of Refmac, that first refinement may
   take as many as 20 cycles to converge, not the default 10 cycles.
   After that, 20 cycles is usually overkill.

Have fun, and solve your protein structure.

Cheers,


___
Roger S. Rowlett
Gordon  Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu


On 3/27/2013 12:22 PM, Tom Van den Bergh wrote:

Dear members of ccp4bb,

I need some help with the refinement of my structure of a variant of 
mRFP (monomer red fluorescent protein, sequence in attachment). I have 
done molecular replacement with phaser with model 2VAD of protein 
database. Then i have done some model building phenix.autobuild. (2 
pdb's (overall...), freeR flags and log file attached) When i refine 
with phenix.refine my structure i get a R-value of 0,42 which is still 
way too high. (redfluorescent protein.pdb, .mtz and logfile attached) 
When i look at the structure in coot i find many unmodelled blobs and 
many outliers in density analysis and rotamer analysis. The problem is 
that there are so many problems with my structure, that i dont know 
where to begin. Could you try some refinement for me, because this is 
first structure that i need to solve as a student and i dont have too 
many experience with it.


Greetings,

Tom

Re: [ccp4bb] refinement protein structure

[Antony Oliver]

Dear Tom, 

I think that we've all actually been rather gently teasing you - admittedly a 
difficult concept to put across via the medium of e-mail.
In fact, I think we've all offered sensible constructive suggestions, and 
indeed pointed out what you should try next.

Apologies for any inadvertent offence - none intended. 

Tony.


 Dear so-far-posters,
 
 I do not know Tom Van den Bergh, nor do I know his background, nor the
 history of the data, nor the reasons why he may have sent it to this
 list (although I think he did it to ask for help), but I find these
 answers irritatingly disrespectful and nasty.

harsh

 No regards to the ones addressed,
 Tim

/harsh

Re: [ccp4bb] refinement protein structure

[Frank von Delft]

Is it too much to dream that Tom has set a trail-blazing precedent and 
demonstrated to us all how unnecessary it is be anal about our 
oh-so-precious data and structures that in the year 2013 are almost 
completely useless without a huge dollop of other experimental data...?




On 27/03/2013 18:32, Anastassis Perrakis wrote:

I think it will be the first time in 15 years I will disagree with Tim.

I personally  found the posting of Tom van der Bergh irritatingly disrespectful 
in many levels.

1. It does not respect my mailbox capacity
2. It does not respect CCP4 developers posting output from phenix.refine
3. It does not respect his supervisors and colleagues who (right now) look like 
fools (to me)
4. It does not respect himself, as I actually suspect he is a proactive 
motivated student who came out as a bit of a fool

These said, I am rather easily irritated these days, so I will not comment on 
the irritable character of the email.

As for the answers, some were funny, some were informative, some funny and 
informative.
Not too much political correctness please, because we will soon start calling 
disordered loops
positionally challenged polypeptide segments (*).

Tassos

(*) joke stolen from Thomas Schneider talk @Stanford, 1998. What a great 
meeting...!


Dear so-far-posters,

I do not know Tom Van den Bergh, nor do I know his background, nor the
history of the data, nor the reasons why he may have sent it to this
list (although I think he did it to ask for help), but I find these
answers irritatingly disrespectful and nasty.

No regards to the ones addressed,
Tim

Re: [ccp4bb] refinement protein structure

[Alexander Aleshin]

But the amount of time spent on turning a protein into a publishable structural 
data is pretty much same, if not larger. There are no low hanging fruits any 
more. 

On Mar 27, 2013, at 11:50 AM, Frank von Delft wrote:

 Is it too much to dream that Tom has set a trail-blazing precedent and 
 demonstrated to us all how unnecessary it is be anal about our oh-so-precious 
 data and structures that in the year 2013 are almost completely useless 
 without a huge dollop of other experimental data...?
 
 
 
 On 27/03/2013 18:32, Anastassis Perrakis wrote:
 I think it will be the first time in 15 years I will disagree with Tim.
 
 I personally  found the posting of Tom van der Bergh irritatingly 
 disrespectful in many levels.
 
 1. It does not respect my mailbox capacity
 2. It does not respect CCP4 developers posting output from phenix.refine
 3. It does not respect his supervisors and colleagues who (right now) look 
 like fools (to me)
 4. It does not respect himself, as I actually suspect he is a proactive 
 motivated student who came out as a bit of a fool
 
 These said, I am rather easily irritated these days, so I will not comment 
 on the irritable character of the email.
 
 As for the answers, some were funny, some were informative, some funny and 
 informative.
 Not too much political correctness please, because we will soon start 
 calling disordered loops
 positionally challenged polypeptide segments (*).
 
 Tassos
 
 (*) joke stolen from Thomas Schneider talk @Stanford, 1998. What a great 
 meeting...!
 
 Dear so-far-posters,
 
 I do not know Tom Van den Bergh, nor do I know his background, nor the
 history of the data, nor the reasons why he may have sent it to this
 list (although I think he did it to ask for help), but I find these
 answers irritatingly disrespectful and nasty.
 
 No regards to the ones addressed,
 Tim

Re: [ccp4bb] refinement protein structure

[Bosch, Juergen]

Hi Tom,

some suggestions for you:

You should calculate a selfrotation function and see if you can learn something 
from it.
You should definitely run Matthews Coefficient and see if you get a brilliant 
idea there

And congratulations to your first crystal structure it looks really great, for 
a starter this is the right resolution to play with and get exposed to all the 
programs used in the crystallographic community.

You got my other email off-the board but for the record keepers here, in case 
you were offended by my early April fools joke I do apologize. And the second 
part in the private email still holds true.

Jürgen

P.S. may I use your data in an X-ray workshop ?

On Mar 27, 2013, at 5:26 PM, Alexander Aleshin wrote:

But the amount of time spent on turning a protein into a publishable structural 
data is pretty much same, if not larger. There are no low hanging fruits any 
more.

On Mar 27, 2013, at 11:50 AM, Frank von Delft wrote:

Is it too much to dream that Tom has set a trail-blazing precedent and 
demonstrated to us all how unnecessary it is be anal about our oh-so-precious 
data and structures that in the year 2013 are almost completely useless without 
a huge dollop of other experimental data...?



On 27/03/2013 18:32, Anastassis Perrakis wrote:
I think it will be the first time in 15 years I will disagree with Tim.

I personally  found the posting of Tom van der Bergh irritatingly disrespectful 
in many levels.

1. It does not respect my mailbox capacity
2. It does not respect CCP4 developers posting output from phenix.refine
3. It does not respect his supervisors and colleagues who (right now) look like 
fools (to me)
4. It does not respect himself, as I actually suspect he is a proactive 
motivated student who came out as a bit of a fool

These said, I am rather easily irritated these days, so I will not comment on 
the irritable character of the email.

As for the answers, some were funny, some were informative, some funny and 
informative.
Not too much political correctness please, because we will soon start calling 
disordered loops
positionally challenged polypeptide segments (*).

Tassos

(*) joke stolen from Thomas Schneider talk @Stanford, 1998. What a great 
meeting...!

Dear so-far-posters,

I do not know Tom Van den Bergh, nor do I know his background, nor the
history of the data, nor the reasons why he may have sent it to this
list (although I think he did it to ask for help), but I find these
answers irritatingly disrespectful and nasty.

No regards to the ones addressed,
Tim

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry  Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://lupo.jhsph.edu

Re: [ccp4bb] refinement of ligands

[Eleanor Dodson]

What do you mean - running REFMAC directly on the output file? 

Are you sure you have the same space group given in the mtz file and the PDB 
file? If the MR has placed your structure in P32 say, nd the input mtz has SG 
P31, you need to change the mtz header to include the now-known SG. There are 
various ways given on the Reflection Utility task in the GUI.
Eleanor
On 30 Jul 2012, at 11:04, Damian Niegowski wrote:

 Dear  all,
 
 I am currently trying to refine co-crystallized  ligand structures at 2.8-3.4 
 Å resolution. After initial molecular replacement the density in the maps, 
 both 2Fo-Fc and Fo-Fc looks good and the ligand seams to have
 bound. However after running Refmac directly on the output files the maps get 
 much worse. I am  using a clean pdb, without ligand or water for the Phaser 
 and subsequent Refmac runs.
 Also when refining with the ligand in the B factors are a lot higher for the 
 ligand then the surrounding residues, only when lowering the occupancy to 
 0.7-0.8 for the ligand the B factors look
 better. Is that acceptable to do at this resolution? R-free is in the 
 0.24-0.27 range. There is only a marginal change in R-free with the addition 
 of ligand. TLS refinement seams to help alot for overall
 R values but does not improve the maps. I have also tried Phenix with 
 simulated annealing, rigid body and reference structures.
 So the question is how best to proceed with refinement at the rather low 
 resolution??
 
 Regards, 
 
 Damian
 
 Damian Niegowski Ph.D.
 Institute of Medical Biochemistry and Biophysics
 Karolinska Institutet
 Scheeles väg 2
 171 77 STOCKHOLM  
 e-mail: damian.niegow...@ki.se
 phone: 0046 8 524 876 33
 fax: 0046 8 736 04 39

Re: [ccp4bb] refinement of ligands

[Herman . Schreuder]

Dear Damian,

The initial map after molecular replacement and BEFORE atomic refinement is 
maybe not the highest quality map, but definitively the map with the least 
model bias. If you see good density for you inhibitors in those maps, they 
almost certainly will have bound. In that case I would fit the inhibitor in 
those very first maps and continue from there.
The inhibitor density probably got worse during refmac refinement without 
inhibitor since refinement programs try to minimize the difference between Fobs 
and Fcalc. If an inhibitor is present in Fobs, but not in the model (Fcalc), 
the refinement program might try to tweak the model such that this difference 
(the inhibitor) gets flattened out. This is called model bias. Modern 
refinement programs try to counteract this effect by using maximum likelyhood 
or other statistical methods, but apparently at the low resolution you have 
refmac did not have enough data to succesfully remove this model bias. You may 
want to try buster for refinement, since this program may suffer less from 
model bias. Buster also allows you to do a group occupancy refinement of you 
inhibitor, since it may indeed by present at partial occupancy.

An Rfree of 0.24 would be ok, 0.27 is a little high but still not really 
worrying. If your Rfactors go down a lot with TLS, this means that you have a 
lot of movement in your protein and that your maps are as good as they will get.

Best,
Herman

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Damian 
Niegowski
Sent: Monday, July 30, 2012 12:04 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] refinement of ligands

Dear  all,

I am currently trying to refine co-crystallized  ligand structures at 2.8-3.4 Å 
resolution. After initial molecular replacement the density in the maps, both 
2Fo-Fc and Fo-Fc looks good and the ligand seams to have bound. However after 
running Refmac directly on the output files the maps get much worse. I am  
using a clean pdb, without ligand or water for the Phaser and subsequent 
Refmac runs.
Also when refining with the ligand in the B factors are a lot higher for the 
ligand then the surrounding residues, only when lowering the occupancy to 
0.7-0.8 for the ligand the B factors look better. Is that acceptable to do at 
this resolution? R-free is in the 0.24-0.27 range. There is only a marginal 
change in R-free with the addition of ligand. TLS refinement seams to help alot 
for overall R values but does not improve the maps. I have also tried Phenix 
with simulated annealing, rigid body and reference structures.
So the question is how best to proceed with refinement at the rather low 
resolution??

Regards, 

Damian

Damian Niegowski Ph.D.
Institute of Medical Biochemistry and Biophysics Karolinska Institutet Scheeles 
väg 2
171 77 STOCKHOLM
e-mail: damian.niegow...@ki.se
phone: 0046 8 524 876 33
fax: 0046 8 736 04 39

Re: [ccp4bb] Refinement with pseudo-translation

[Eleanor Dodson]

You don't say how many molecules you are looking for?

I would try to first work in SG C2 pretending that the pseudo translation 
is 100% of the origin.


Reindex the data as k,l,h, change the SG to C2 and then redo the scaling 
and merging. You will lose all the reflections k+l =2n+1 in your indexing - 
h+k = 2n+1 after reindexing, but the MR should be simpler.


Once you have a C2 solution you can convert it back to the orthorhombic one 
with a certain amount of intellectual struggle! The C2 origin along your b 
axis is free, so you will have to


Or you can use ZANUDU from the ysbl software web site which will maybe help.

Eleannor


On Mar 22 2012, Shiva Kumar wrote:


Dear CCP4bb members

I have a 3.0 Å dataset which has an off-origin peak of height 36% in 
‎patterson map. The peak is at fractional co-ordinates 0, 0.5, 0.5. Data 
has been indexed in P2(1)22(1) SG using HKL2000. I have located all the 
molecules in asu (as far as I know) using Molrep with the 'locked 
rotation' and 'Use' PST feature. After 1 round (20 cycles) of rigid body 
and 1 round (10 cycles) of restrained refinement (Refmac), the R and 
Rfree are 49 and 53 %. Although the R factors are very high, I feel the 
solution might be correct because the electron density follows c-alpha 
trace in almost all places. To be sure, I deleted a beta strand from the 
structure's core and repeated the refinement and found that the electron 
density for the strand was still present (I have no experimental phases). 
I have the following questions:


1) Are the final R factors never expected to reduce to acceptable values 
given the 36% off-origin peak?


2) What is the best way to settle the SG? I was considering 
P2(1)2(1)2(1), P2(1)22, P2(1)2(1)2 and P2(1)22(1), considering the 
off-origin peak is at 0,0.5,0.5. I found all the molecules in asu only 
using the P2(1)22(1) data in Molrep. Is this the best way to settle my 
SG?


3) Some CCP4bb archives advise either refining against weak and strong 
reflections alternatively 
(https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=ind1010L=ccp4bbD=01=ccp4bb9=AJ=ond=No+Match%3BMatch%3BMatchesz=4P=204490) 
or refining against medium intensity reflections. Should I also be doing 
these things? If yes, then what is the best way of doing it?


Your suggestions and corrections to my interpretation of our data would 
be appreciated.


Regards
Shiva



--
Professor Eleanor Dodson
YSNL, Dept of Chemistry
University of York
Heslington YO10 5YW
tel: 00 44 1904 328259
Fax: 00 44 1904 328266

Re: [ccp4bb] Refinement with pseudo-translation

[Boaz Shaanan]

Hi,

I would go even further down SG ladder, i.e. all the way to P1, reprocess the 
data in P1 (you won't lose reflections with special indices that way, the only 
thing to worry here is completeness, but hopefully you recorded highly 
redundant data) and do the MR. It worked for me and for others.

  Cheers,

 Boaz


Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Israel

E-mail: bshaa...@bgu.ac.il
Phone: 972-8-647-2220  Skype: boaz.shaanan
Fax:   972-8-647-2992 or 972-8-646-1710






From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Eleanor Dodson 
[eleanor.dod...@york.ac.uk]
Sent: Sunday, March 25, 2012 9:55 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Refinement with pseudo-translation

You don't say how many molecules you are looking for?

I would try to first work in SG C2 pretending that the pseudo translation
is 100% of the origin.

Reindex the data as k,l,h, change the SG to C2 and then redo the scaling
and merging. You will lose all the reflections k+l =2n+1 in your indexing -
h+k = 2n+1 after reindexing, but the MR should be simpler.

Once you have a C2 solution you can convert it back to the orthorhombic one
with a certain amount of intellectual struggle! The C2 origin along your b
axis is free, so you will have to

Or you can use ZANUDU from the ysbl software web site which will maybe help.

Eleannor


On Mar 22 2012, Shiva Kumar wrote:

Dear CCP4bb members

 I have a 3.0 Å dataset which has an off-origin peak of height 36% in
 ‎patterson map. The peak is at fractional co-ordinates 0, 0.5, 0.5. Data
 has been indexed in P2(1)22(1) SG using HKL2000. I have located all the
 molecules in asu (as far as I know) using Molrep with the 'locked
 rotation' and 'Use' PST feature. After 1 round (20 cycles) of rigid body
 and 1 round (10 cycles) of restrained refinement (Refmac), the R and
 Rfree are 49 and 53 %. Although the R factors are very high, I feel the
 solution might be correct because the electron density follows c-alpha
 trace in almost all places. To be sure, I deleted a beta strand from the
 structure's core and repeated the refinement and found that the electron
 density for the strand was still present (I have no experimental phases).
 I have the following questions:

 1) Are the final R factors never expected to reduce to acceptable values
 given the 36% off-origin peak?

 2) What is the best way to settle the SG? I was considering
 P2(1)2(1)2(1), P2(1)22, P2(1)2(1)2 and P2(1)22(1), considering the
 off-origin peak is at 0,0.5,0.5. I found all the molecules in asu only
 using the P2(1)22(1) data in Molrep. Is this the best way to settle my
 SG?

 3) Some CCP4bb archives advise either refining against weak and strong
 reflections alternatively
 (https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=ind1010L=ccp4bbD=01=ccp4bb9=AJ=ond=No+Match%3BMatch%3BMatchesz=4P=204490)
 or refining against medium intensity reflections. Should I also be doing
 these things? If yes, then what is the best way of doing it?

 Your suggestions and corrections to my interpretation of our data would
 be appreciated.

Regards
Shiva


--
Professor Eleanor Dodson
YSNL, Dept of Chemistry
University of York
Heslington YO10 5YW
tel: 00 44 1904 328259
Fax: 00 44 1904 328266