[ccp4bb] add ligand solution onto drop directly SUMMARY

[Markus Heckmann]

Thanks for the responses. Yes, my crystal did survive and I can see
density for my ligand.

Summary:
1. If the crystal survives, it’s fine. These are just different ways
of exposing the crystal to the ligand and do what works. The only
issue will be if you don’t see electron density for the ligand in your
structure. If you just add ligand to the drop then there is plenty of
other precipitate/stuff that could non-specifically bind to the ligand
and reduce the amount available to bind the protein in the crystal. So
if you don’t see density, it would still be worth soaking a “clean”
crystal in the ligand/precipitant solution.

2.  It is OK as long as your crystal survives. I do this regularly, or
even just add dry compound to the drops directly, usually after adding
more reservoir to make the drop a bit bigger. It seems to work fine
for some compounds, not for others. It is very empirical.

3. The truth is in the map. Ergo, zap it and rationalize why it worked
or not later.

Re: [ccp4bb] CCP4BB Digest - 24 Jan 2017 to 25 Jan 2017 (#2017-26)

[Ethan Merritt]

On Thursday, 26 January 2017 11:03:12 PM Claire Smith wrote:
> Hello,
> 
> I would like to ask a question related to a recent thread regarding
> bad/missing density.
> 
> I have used SAD to solve the structure of a protein at about 2.6Å
> resolution. Phenix build a good portion of it (about 50%) and the density
> in this region is good. However, we cannot see ther rest 50%.  Rfree is
> currently at 32%.  No twinning is suggested.
> 
> How can we "find" the missing 50%? We have tried MR-SAD with no significant
> improvement. We know the missing mass is there because we ran the crystals
> on a  gel, and the protein is intact.
> 
> I know with MR sometimes the space group can make a difference, but with
> experimental phasing (SAD),  if the space group were incorrectly
> identified, would we have gotten a solution to the Se substructure? This
> seems correct because the visible 50% of the protein make total sense. So,
> since we have a correct substructure, can I conclude with confidence that
> the space group is correctly identified?

>From your description it sounds unlikely that the unit cell is correct but
the space group is wrong.   However the symptoms do match a possible
missed supercell, such that you collected and refined data from only 
a subset of the true unique data.

I will try to describe this in words but a picture would be so much easier...
I will try ascii art.  Please view in a fixed spacing font.
Suppose the true cell looks like this:
---
| AAA|  AAA  BBB  |
| B  |  BBBB  |
| AAA   BBB  |  AAA   |
---

I.e. suppose you have pseudo-translational NCS such that domain A
superimposes perfectly on itself but domain B does not.
If you incorrectly index that cell edge as being only 1/2 its true length,
you measure only half of the true data but it refines nicely to 
describe a fully-occupied A and a mess in the region where B should be.
(superimposed 1/2 intensity ghosts made noisier by the missing data).

Yes I've hit this in real life, with an even messier case of cell-edge
tripling rather than doubling.
If you want to pursue this possibility, you should go back to the
diffraction images and look really hard for weak spots in between the
indexed spots.

good luck,

Ethan

 
> Of course, it could be that the missing bit is very flexible and does not
> scatter coherently, but then, wouldn't we expect a lower Rfree?

An Rfree of 0.32 for 2.6A refinement doesn't sound that bad.

 
> Thanks so much!
> 
> Claire
> 

-- 
Ethan A Merritt, Dept of Biochemistry
Biomolecular Structure Center,  K-428 Health Sciences Bldg
MS 357742,   University of Washington, Seattle 98195-7742

Re: [ccp4bb] CCP4BB Digest - 24 Jan 2017 to 25 Jan 2017 (#2017-26)

[Claire Smith]

Hello,

I would like to ask a question related to a recent thread regarding
bad/missing density.

I have used SAD to solve the structure of a protein at about 2.6Å
resolution. Phenix build a good portion of it (about 50%) and the density
in this region is good. However, we cannot see ther rest 50%.  Rfree is
currently at 32%.  No twinning is suggested.

How can we "find" the missing 50%? We have tried MR-SAD with no significant
improvement. We know the missing mass is there because we ran the crystals
on a  gel, and the protein is intact.

I know with MR sometimes the space group can make a difference, but with
experimental phasing (SAD),  if the space group were incorrectly
identified, would we have gotten a solution to the Se substructure? This
seems correct because the visible 50% of the protein make total sense. So,
since we have a correct substructure, can I conclude with confidence that
the space group is correctly identified?

Of course, it could be that the missing bit is very flexible and does not
scatter coherently, but then, wouldn't we expect a lower Rfree?

Thanks so much!

Claire






On Wed, Jan 25, 2017 at 7:00 PM, CCP4BB automatic digest system <
lists...@jiscmail.ac.uk> wrote:

> There are 13 messages totaling 4586 lines in this issue.
>
> Topics of the day:
>
>   1. add ligand solution onto drop directly (2)
>   2. Bad density for chains (3)
>   3. Unknown electron density blob (2)
>   4. Macromolecular Crystallography School MCS2017 Madrid
>   5. explain "comfortable"
>   6. Unknown electron density blob, pdb convention for partially ordered
>  ligands (3)
>   7. PhD Presidential fellowship to study the Structure and function of
> CRISPR
>  systems
>
> --
>
> Date:Wed, 25 Jan 2017 03:23:18 +0100
> From:Markus Heckmann 
> Subject: add ligand solution onto drop directly
>
> Dear all,
> I wondered if it is OK to pipette ligand soln (X-CoA) *directly* to the
> drop with crystal (1:1 ratio of protein:precipitant 2µl) instead of
> dissolving it in precipitant solution and transferring the crystal to this
> ligand containing precipitant solution. The crystals survive this as I add
> the ligand solution to the edge of the drop and gently mix the two
> solutions. Since I collected my datasets I wonder if it is OK?
>
> Many thanks,
> Markus
>
> --
>
> Date:Tue, 24 Jan 2017 19:15:43 -0800
> From:Bernhard Rupp 
> Subject: Re: add ligand solution onto drop directly
>
> The truth is in the map. Ergo, zap it and rationalize why it worked or not
> later.
>
>
>
> Cheers, BR
>
>
>
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
> Markus Heckmann
> Sent: Tuesday, January 24, 2017 6:23 PM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] add ligand solution onto drop directly
>
>
>
> Dear all,
>
> I wondered if it is OK to pipette ligand soln (X-CoA) *directly* to the
> drop with crystal (1:1 ratio of protein:precipitant 2µl) instead of
> dissolving it in precipitant solution and transferring the crystal to this
> ligand containing precipitant solution. The crystals survive this as I add
> the ligand solution to the edge of the drop and gently mix the two
> solutions. Since I collected my datasets I wonder if it is OK?
>
> Many thanks,
>
> Markus
>
> --
>
> Date:Wed, 25 Jan 2017 10:12:55 +0530
> From:Pooja Kesari 
> Subject: Bad density for chains
>
> Dear All,
>
> I have a 2.6 A resolution structure having four chains in an asymmetric
> unit.
> The chain A and B have density for almost all residues however we don't
> have proper residue density in chain C and D.What can be tried to build
> chain C and D ?
>
>
>
> Many Thanks
> Pooja
>
> --
>
> Date:Tue, 24 Jan 2017 21:02:40 -0800
> From:Debanu 
> Subject: Re: Bad density for chains
>
> Hi Pooja,
>
> Are you positive you have the correct space group and there are no other
> issues like twinning, etc?
>
> If sure, did you define NCS groups in refinement? TLS refinement? Try
> different refinement programs?
>
> How big is the molecule? Was it solved by MR or experimental phasing?
>
> You can try superimposing A/B on C/D and refinement with tight NCS then
> adjust NCS restraints during model adjustments based on local differences
> or also see if phenix autobuild helps.
>
> Best,
> Debanu
> --
> Debanu Das
> Accelero Biostructures
>
>
> > On Jan 24, 2017, at 8:42 PM, Pooja Kesari  wrote:
> >
> > Dear All,
> >
> > I have a 2.6 A resolution structure having four chains in an asymmetric
> unit.
> > The chain A and B have density for almost all residues however we don't
> have proper residue density in chain C and D.What can be tried to build
> chain C and D ?
> >
> >
> >
> > Many Thanks
> > Pooja
>
> --
>
> 

[ccp4bb] TLSMD server down with disk problems

[Ethan A Merritt]

The TLSMD server is suffering from hardware problems.
It is unclear how long it will take to fix this, or whether
the whole machine will need to be replaced.

I'd love it if someone would undertake to run a mirror
or equivalent server, but as it stands I think you're out of luck
until I can round up enough resources to fix/repair/replace mine.

sorry,

Ethan


On Thursday, 26 January, 2017 21:36:51 Carlos CONTRERAS-MARTEL wrote:
> Hi CCP4BB,
> 
> 
> Somebody know what happen with the TLSDM server at 
> http://skuld.bmsc.washington.edu/~tlsmd/
> 
> It has been refusing connections for already two days?
> 
> Perhaps a mirror exists somewhere ?
> 
> 
> Best
> 
> 
> Carlos
> 
> 
> 

-- 
Ethan A Merritt
Biomolecular Structure Center,  K-428 Health Sciences Bldg
MS 357742,   University of Washington, Seattle 98195-7742

[ccp4bb] Experimental Postdoctoral Position in High Throughput Small Molecule Ligand Screening

[Jessica Forness]

Outstanding postdoctoral applicants are sought with the following 
qualifications to work with Dr. Jeffrey Skolnick in the Center for the Study of 
Systems Biology in the School of Biological Sciences at the Georgia Institute 
of Technology:

• Extensive experience in enzyme kinetics studies, enzyme purification 
or other aspects of protein biology and enzyme activity. Experience in handling 
multiple protein systems would be a plus.

• A background in high throughput small molecule ligand screening is 
strongly preferred.

• Experience with or a desire to learn computational biology and 
molecular modeling of protein-ligand interactions.

• The ideal candidate is someone who gets satisfaction out of methods 
development and working through large data sets to see broad-scale patterns.

• A track record of publication in high quality, international journals.

• Ability to work in a team, yet think independently.

To apply, please email your CV to: skoln...@gatech.edu

[ccp4bb] TLSMD Server ?

[Carlos CONTRERAS-MARTEL]

Hi CCP4BB,


Somebody know what happen with the TLSDM server at 
http://skuld.bmsc.washington.edu/~tlsmd/


It has been refusing connections for already two days?

Perhaps a mirror exists somewhere ?


Best


Carlos


--
 Carlos CONTRERAS MARTEL, Ph.D.
 (CR1 CNRS)

 carlos.contreras-mar...@ibs.fr

 "Bacterial Pathogenesis Group"
Institut de Biologie Structurale
 UMR5075 CEA-CNRS-UGA

  IBS
  Campus EPN
  71, avenue des Martyrs
  CS 10090
  38044 Grenoble CEDEX 9
  FRANCE


 tel : (+33) (0)4 57 42 86 41

http://www.ibs.fr/groupes/groupe-pathogenie-bacterienne/?lang=fr
http://www.ibs.fr/groups/bacterial-pathogenesis-group/?lang=en

[ccp4bb] 5th Banff Meeting on Structural Dynamics, 19th Febr. - 22nd Febr. 2017 / approaching deadline for late abstract submission for posters

[Kleine, Irmtraud]

Dear all,

 

Please mind the approaching deadline  (= 30th January 2017) for abstract
submission regarding posters to be presented at the 5th Banff Meeting on
Structural Dynamics https://banff2017.desy.de/  

 

Best regards,

 

 

Irmtraud Kleine

Assistant to Professor Chapman

Deutsches Elektronen-Synchrotron DESY

Ein Forschungszentrum der Helmholtz-Gemeinschaft

Center for Free-Electron Laser Science (FS-CFEL-1)

Notkestr. 85

22607 Hamburg

Germany

 

Phone:  +49 40 8998 5798

Fax:+49 40 8994 5798   

E-mail:   irmtraud.kle...@desy.de   

 

 

 

 

 

 

Re: [ccp4bb] wavelength dependence of intensities

[Loes Kroon-Batenburg]

Dear Tim,

That is described by the Laue interference function. The crystal 
diffracts in Bragg directions (reflections) at small deviations of the 
Bragg angle and in a small solid angle. They depend on lambda and 
lambda^2 respectively, and integration over these angles brings a factor 
lambda^3 in the diffracted intensity.


Best wishes,
Loes
On 01/26/17 17:15, Tim Gruene wrote:

Dear all,

according to Carmelo Giacovazzo's textbook (eq. 3.41 in the 1st edition), the
diffraction intensity I(hkl) scales with the wavelength cubed (lambda^3).

1) is there an easy rationale for this dependency?
2) does it hold over a wide range of energies?
3) does this also hold for neutrons or electrons as radiation source?

Thanks a lot for any helpful comment,
Tim



--

__

Dr. Loes Kroon-Batenburg
Dept. of Crystal and Structural Chemistry
Bijvoet Center for Biomolecular Research
Utrecht University
Padualaan 8, 3584 CH Utrecht
The Netherlands

E-mail : l.m.j.kroon-batenb...@uu.nl
phone  : +31-30-2532865
fax: +31-30-2533940
__

Re: [ccp4bb] glycosylated density blob?

[Zachary A. Wood]

You mentioned that you have PMSF in your crystallization condition…and PMSF 
does modify serines.

Best regards,

Z


***
Zachary A. Wood, Ph.D.
Associate Professor  
Department of Biochemistry & Molecular Biology
University of Georgia
Life Sciences Building, Rm A426B
120 Green Street
Athens, GA  30602-7229
Office: 706-583-0304
Lab:706-583-0303
FAX: 706-542-1738
***



> On Jan 26, 2017, at 11:13 AM, Mark J van Raaij  wrote:
> 
> well yes, looks like a glycosylation to me too.
> However, at 2.75Å resolution I think it will be impossible to know which one 
> from the density alone.
> Perhaps mass spectroscopy can give a clue?
> Or get clues from what is known about your protein and your expression system.
> 
> Mark J van Raaij
> Dpto de Estructura de Macromoleculas
> Centro Nacional de Biotecnologia - CSIC
> calle Darwin 3
> E-28049 Madrid, Spain
> tel. (+34) 91 585 4616
> http://wwwuser.cnb.csic.es/~mjvanraaij
> 
> 
> 
> 
> 
> 
> 
>> On 26 Jan 2017, at 15:45, KH. HEROJIT SINGH, PH D SCHOLAR, PCL 
>>  wrote:
>> 
>> Dear All,
>> 
>> 
>> A continuous map from gamma O of serine at expose region was observed. 
>> Resolution of dataset is 2.75 A. The serine position is predicted for 
>> phosphorylation in-sillico. With fitting with mannose, density is not fully 
>> satisfied while with GalNac, some negative density is found. 
>> 
>> 
>> Condition was 15% PEG 3350, Tris 50mM pH 7, 100mM Na2SO4and buffer was  tris 
>> 50mM, NaCl 50mM, ph 8.5 , 1mM PMSF. Images from different angle are attached 
>> (File size is 300Kb). Arrow in fig pointed the same serine residue.
>> 
>> 
>> Kindly suggest.  
>> 
>> -- 
>> Regards,
>> KHUNDRAKPAM HEROJIT 
>> Ph D SCHOLAR
>> PROTEIN CRYSTALLOGRAPHY LAB.
>> NATIONAL INSTITUTE OF IMMUNOLOGY
>> NEW DELHI-110067, INDIA

[ccp4bb] wavelength dependence of intensities

[Tim Gruene]

Dear all,

according to Carmelo Giacovazzo's textbook (eq. 3.41 in the 1st edition), the 
diffraction intensity I(hkl) scales with the wavelength cubed (lambda^3).

1) is there an easy rationale for this dependency?
2) does it hold over a wide range of energies?
3) does this also hold for neutrons or electrons as radiation source?

Thanks a lot for any helpful comment,
Tim
-- 
--
Paul Scherrer Institut
Dr. Tim Gruene
- persoenlich -
Principal Investigator
Biology and Chemistry
OFLC/102
CH-5232 Villigen PSI

Phone: +41 (0)56 310 5297

GPG Key ID = A46BEE1A



signature.asc
Description: This is a digitally signed message part.

Re: [ccp4bb] glycosylated density blob?

[Mark J van Raaij]

well yes, looks like a glycosylation to me too.
However, at 2.75Å resolution I think it will be impossible to know which one 
from the density alone.
Perhaps mass spectroscopy can give a clue?
Or get clues from what is known about your protein and your expression system.

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser.cnb.csic.es/~mjvanraaij







> On 26 Jan 2017, at 15:45, KH. HEROJIT SINGH, PH D SCHOLAR, PCL 
>  wrote:
> 
> Dear All,
> 
>  
> A continuous map from gamma O of serine at expose region was observed. 
> Resolution of dataset is 2.75 A. The serine position is predicted for 
> phosphorylation in-sillico. With fitting with mannose, density is not fully 
> satisfied while with GalNac, some negative density is found. 
> 
>  
> Condition was 15% PEG 3350, Tris 50mM pH 7, 100mM Na2SO4and buffer was  tris 
> 50mM, NaCl 50mM, ph 8.5 , 1mM PMSF. Images from different angle are attached 
> (File size is 300Kb). Arrow in fig pointed the same serine residue.
> 
> 
> Kindly suggest.  
> 
> -- 
> Regards,
> KHUNDRAKPAM HEROJIT 
> Ph D SCHOLAR
> PROTEIN CRYSTALLOGRAPHY LAB.
> NATIONAL INSTITUTE OF IMMUNOLOGY
> NEW DELHI-110067, INDIA

Re: [ccp4bb] Bad density for chains

[benjamin bax]

You could try the diffraction anisotropy server: 
http://services.mbi.ucla.edu/anisoscale/.
Sometimes it helps maps become more interpretable. 

 Ben  


On 26 Jan 2017, at 14:11, Pooja Kesari  wrote:

We have a 2.6 A structure showing four chains in an asymmetric unit. Our 
protein is 360 residues around 40 kDa . Mattews shows four chain in an 
assymetric unit (solvent 49% mattews coeff 2.44). The template has about 60% 
homologous with our protein. The molecular replacement against this template 
gave an initial free R of 38.  We did chain tracing and found that we have good 
density (2Fo-Fc) for chain A and B but poor density for C and D. 

1. The density for a particular stretch of 10 amino acids (disordered loop 
region) is absent in all the chains. We could not found density for this 
flexible loop region in any of the already known structures. Any suggestion on 
how can we build this region?

2. We did not find density for most of the loop regions in chain C and D which 
were well traced in chain A and B. How can we improving the density for these 
two chains based on chain A and B (Density modification)? 

3. We analysed the data using phenix xtriage and found that our data shows 
severe anisotropy. Any suggestion of anisotropy correction?

Pointless and Ctruncate analyses didn't show twinning or NCS.  I have checked 
the space group using Zanuda. We are stuck at a free value of 32. 

On Thu, Jan 26, 2017 at 4:47 PM, Eleanor Dodson > wrote:
This is a bit too vague to help much.
How did you solve the structure?
Eleanor

On 26 January 2017 at 03:50, Pooja Kesari > wrote:
Dear All,
Thank you all for reply.

We have checked the data for twinning.
Our protein is 360 residues around 40 kDa protein.
We have tried TLS refinement.
chain A and B don't superimpose well with chain C and D. (A and B chains also 
share slight difference )
Since we don't have proper density for some regions  chain C and D, we are not 
sure whether these chain have similar or different conformations. 
We tried anisotropy correction and the model refined a bit.


On Wed, Jan 25, 2017 at 10:32 AM, Debanu > wrote:
Hi Pooja,

Are you positive you have the correct space group and there are no other issues 
like twinning, etc?

If sure, did you define NCS groups in refinement? TLS refinement? Try different 
refinement programs?

How big is the molecule? Was it solved by MR or experimental phasing?

You can try superimposing A/B on C/D and refinement with tight NCS then adjust 
NCS restraints during model adjustments based on local differences or also see 
if phenix autobuild helps. 

Best,
Debanu 
--
Debanu Das
Accelero Biostructures 


On Jan 24, 2017, at 8:42 PM, Pooja Kesari > wrote:

> Dear All,
> 
> I have a 2.6 A resolution structure having four chains in an asymmetric unit.
> The chain A and B have density for almost all residues however we don't have 
> proper residue density in chain C and D.What can be tried to build chain C 
> and D ?
> 
> 
> 
> Many Thanks 
> Pooja



-- 
Thanks & Regards,
Pooja Kesari
Research Scholar
Department Of Biotechnology
Indian Institute of Technology Roorkee
INDIA





-- 
Thanks & Regards,
Pooja Kesari
Research Scholar
Department Of Biotechnology
Indian Institute of Technology Roorkee
INDIA



Dr Ben Bax

Dept. Biological Chemistry, 
John Innes Centre, 
Norwich Research Park, 
Norwich NR4 7UH, UK

ben.d.v@gmail.com

 

 

[ccp4bb] AW: [ccp4bb] Bad density for chains

[Herman . Schreuder]

Dear Pooja,

A few remarks:


-Matthews does not show 4 chains in the asymmetric unit, it suggests 4 
chains. However, in reality it can be more or less chains, although rare, 75% 
solvent (2 chains) is not unheard of.

-An initial Rfree of 38% is ok, 32% after refinement is a bit high

-Disordered loops do exist and you may have to live with them (not be 
able to build them).

-To correct for anisotropy, I suggest the staraniso server from global 
phasing.

-Unless you ran your molecular replacement in P1, Zanuda will confirm 
the space group you choose for MR. So run MR in P1 (if your symmetry is not too 
high) and run Zanuda again. (Have someone) look critical at the assigned space 
group and assess whether another choice might also be possible.

Good luck!
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Pooja 
Kesari
Gesendet: Donnerstag, 26. Januar 2017 15:12
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Bad density for chains

We have a 2.6 A structure showing four chains in an asymmetric unit. Our 
protein is 360 residues around 40 kDa . Mattews shows four chain in an 
assymetric unit (solvent 49% mattews coeff 2.44). The template has about 60% 
homologous with our protein. The molecular replacement against this template 
gave an initial free R of 38.  We did chain tracing and found that we have good 
density (2Fo-Fc) for chain A and B but poor density for C and D.

1. The density for a particular stretch of 10 amino acids (disordered loop 
region) is absent in all the chains. We could not found density for this 
flexible loop region in any of the already known structures. Any suggestion on 
how can we build this region?

2. We did not find density for most of the loop regions in chain C and D which 
were well traced in chain A and B. How can we improving the density for these 
two chains based on chain A and B (Density modification)?

3. We analysed the data using phenix xtriage and found that our data shows 
severe anisotropy. Any suggestion of anisotropy correction?

Pointless and Ctruncate analyses didn't show twinning or NCS.  I have checked 
the space group using Zanuda. We are stuck at a free value of 32.

On Thu, Jan 26, 2017 at 4:47 PM, Eleanor Dodson 
> wrote:
This is a bit too vague to help much.
How did you solve the structure?
Eleanor

On 26 January 2017 at 03:50, Pooja Kesari 
> wrote:
Dear All,
Thank you all for reply.
We have checked the data for twinning.
Our protein is 360 residues around 40 kDa protein.
We have tried TLS refinement.
chain A and B don't superimpose well with chain C and D. (A and B chains also 
share slight difference )
Since we don't have proper density for some regions  chain C and D, we are not 
sure whether these chain have similar or different conformations.
We tried anisotropy correction and the model refined a bit.


On Wed, Jan 25, 2017 at 10:32 AM, Debanu 
> wrote:
Hi Pooja,

Are you positive you have the correct space group and there are no other issues 
like twinning, etc?

If sure, did you define NCS groups in refinement? TLS refinement? Try different 
refinement programs?

How big is the molecule? Was it solved by MR or experimental phasing?

You can try superimposing A/B on C/D and refinement with tight NCS then adjust 
NCS restraints during model adjustments based on local differences or also see 
if phenix autobuild helps.

Best,
Debanu
--
Debanu Das
Accelero Biostructures


On Jan 24, 2017, at 8:42 PM, Pooja Kesari 
> wrote:
Dear All,
I have a 2.6 A resolution structure having four chains in an asymmetric unit.
The chain A and B have density for almost all residues however we don't have 
proper residue density in chain C and D.What can be tried to build chain C and 
D ?


Many Thanks
Pooja



--
Thanks & Regards,
Pooja Kesari
Research Scholar
Department Of Biotechnology
Indian Institute of Technology Roorkee
INDIA





--
Thanks & Regards,
Pooja Kesari
Research Scholar
Department Of Biotechnology
Indian Institute of Technology Roorkee
INDIA

Re: [ccp4bb] Bad density for chains

[Eleanor Dodson]

Well - first
Solvent content..

1) there could be 4 molecules, or 3, or 2.. Solvent content varies a lot as
you know. Is there reasonable density for parts of all the chains?

2) Does the MR search clearly show 4 molecules? ie improvement in scores
with extra molecules?

3) Have you checked for sensible NC symmetry - submit your model to the
PISA server and see if it suggests a teramer, or 4 fold symmetry or maybe 2
dimers?
PISA will also suggest buried surface area of each chain - maybe parts of
the C & D chains have nothing to fix them in place
Eleanor



On 26 January 2017 at 14:11, Pooja Kesari  wrote:

> We have a 2.6 A structure showing four chains in an asymmetric unit. Our
> protein is 360 residues around 40 kDa . Mattews shows four chain in an
> assymetric unit (solvent 49% mattews coeff 2.44). The template has about
> 60% homologous with our protein. The molecular replacement against this
> template gave an initial free R of 38.  We did chain tracing and found that
> we have good density (2Fo-Fc) for chain A and B but poor density for C and
> D.
>
> 1. The density for a particular stretch of 10 amino acids (disordered loop
> region) is absent in all the chains. We could not found density for this
> flexible loop region in any of the already known structures. Any suggestion
> on how can we build this region?
>
> 2. We did not find density for most of the loop regions in chain C and D
> which were well traced in chain A and B. How can we improving the density
> for these two chains based on chain A and B (Density modification)?
>
> 3. We analysed the data using phenix xtriage and found that our data shows
> severe anisotropy. Any suggestion of anisotropy correction?
>
> Pointless and Ctruncate analyses didn't show twinning or NCS.  I have
> checked the space group using Zanuda. We are stuck at a free value of 32.
>
> On Thu, Jan 26, 2017 at 4:47 PM, Eleanor Dodson  > wrote:
>
>> This is a bit too vague to help much.
>> How did you solve the structure?
>> Eleanor
>>
>> On 26 January 2017 at 03:50, Pooja Kesari  wrote:
>>
>>> Dear All,
>>> Thank you all for reply.
>>>
>>> We have checked the data for twinning.
>>> Our protein is 360 residues around 40 kDa protein.
>>> We have tried TLS refinement.
>>> chain A and B don't superimpose well with chain C and D. (A and B chains
>>> also share slight difference )
>>> Since we don't have proper density for *some regions*  chain C and D,
>>> we are not sure whether these chain have similar or different
>>> conformations.
>>> We tried anisotropy correction and the model refined a bit.
>>>
>>>
>>> On Wed, Jan 25, 2017 at 10:32 AM, Debanu  wrote:
>>>
 Hi Pooja,

 Are you positive you have the correct space group and there are no
 other issues like twinning, etc?

 If sure, did you define NCS groups in refinement? TLS refinement? Try
 different refinement programs?

 How big is the molecule? Was it solved by MR or experimental phasing?

 You can try superimposing A/B on C/D and refinement with tight NCS then
 adjust NCS restraints during model adjustments based on local differences
 or also see if phenix autobuild helps.

 Best,
 Debanu
 --
 Debanu Das
 Accelero Biostructures


 On Jan 24, 2017, at 8:42 PM, Pooja Kesari  wrote:

 Dear All,

 I have a 2.6 A resolution structure having four chains in an asymmetric
 unit.
 The chain A and B have density for almost all residues however we don't
 have proper residue density in chain C and D.What can be tried to build
 chain C and D ?



 Many Thanks
 Pooja


>>>
>>>
>>> --
>>> Thanks & Regards,
>>> Pooja Kesari
>>> Research Scholar
>>> Department Of Biotechnology
>>> Indian Institute of Technology Roorkee
>>> INDIA
>>>
>>>
>>
>
>
> --
> Thanks & Regards,
> Pooja Kesari
> Research Scholar
> Department Of Biotechnology
> Indian Institute of Technology Roorkee
> INDIA
>
>

Re: [ccp4bb] Bad density for chains

[Pavel Afonine]

Hi,

assuming you've exhausted modeling choices and applied proper refinement
strategies, after all Rree=32% is not unheard of at 2.6A resolution (which
actually may be even worse than 2.6A if data set is not 100% complete).
Have a look at R-factors of models in PDB with data resolution around 2.6A:

Histogram of Rwork for models in PDB at resolution 2.50-2.70 A:
 0.119 - 0.145  : 10
 0.145 - 0.171  : 147
 0.171 - 0.197  : 814
 0.197 - 0.223  : 1454
 0.223 - 0.248  : 951
 0.248 - 0.274  : 183
 0.274 - 0.300  : 31
 0.300 - 0.326  : 3
 0.326 - 0.352  : 0
 0.352 - 0.378  : 1
Histogram of Rfree for models in PDB at resolution 2.50-2.70 A:
 0.175 - 0.204  : 51
 0.204 - 0.233  : 420
 0.233 - 0.262  : 1328
 0.262 - 0.291  : 1344
 0.291 - 0.320  : 386
 0.320 - 0.349  : 58
 0.349 - 0.378  : 6
 0.378 - 0.407  : 0
 0.407 - 0.436  : 0
 0.436 - 0.465  : 1
Histogram of Rfree-Rwork for all model in PDB at resolution 2.50-2.70 A:
 0.002 - 0.012  : 27
 0.012 - 0.022  : 137
 0.022 - 0.031  : 360
 0.031 - 0.041  : 645
 0.041 - 0.051  : 750
 0.051 - 0.061  : 754
 0.061 - 0.071  : 498
 0.071 - 0.080  : 251
 0.080 - 0.090  : 114
 0.090 - 0.100  : 58
Number of structures considered: 3594

Sounds like you are not alone...

Pavel


On Thu, Jan 26, 2017 at 6:11 AM, Pooja Kesari  wrote:

> We have a 2.6 A structure showing four chains in an asymmetric unit. Our
> protein is 360 residues around 40 kDa . Mattews shows four chain in an
> assymetric unit (solvent 49% mattews coeff 2.44). The template has about
> 60% homologous with our protein. The molecular replacement against this
> template gave an initial free R of 38.  We did chain tracing and found that
> we have good density (2Fo-Fc) for chain A and B but poor density for C and
> D.
>
> 1. The density for a particular stretch of 10 amino acids (disordered loop
> region) is absent in all the chains. We could not found density for this
> flexible loop region in any of the already known structures. Any suggestion
> on how can we build this region?
>
> 2. We did not find density for most of the loop regions in chain C and D
> which were well traced in chain A and B. How can we improving the density
> for these two chains based on chain A and B (Density modification)?
>
> 3. We analysed the data using phenix xtriage and found that our data shows
> severe anisotropy. Any suggestion of anisotropy correction?
>
> Pointless and Ctruncate analyses didn't show twinning or NCS.  I have
> checked the space group using Zanuda. We are stuck at a free value of 32.
>
> On Thu, Jan 26, 2017 at 4:47 PM, Eleanor Dodson  > wrote:
>
>> This is a bit too vague to help much.
>> How did you solve the structure?
>> Eleanor
>>
>> On 26 January 2017 at 03:50, Pooja Kesari  wrote:
>>
>>> Dear All,
>>> Thank you all for reply.
>>>
>>> We have checked the data for twinning.
>>> Our protein is 360 residues around 40 kDa protein.
>>> We have tried TLS refinement.
>>> chain A and B don't superimpose well with chain C and D. (A and B chains
>>> also share slight difference )
>>> Since we don't have proper density for *some regions*  chain C and D,
>>> we are not sure whether these chain have similar or different
>>> conformations.
>>> We tried anisotropy correction and the model refined a bit.
>>>
>>>
>>> On Wed, Jan 25, 2017 at 10:32 AM, Debanu  wrote:
>>>
 Hi Pooja,

 Are you positive you have the correct space group and there are no
 other issues like twinning, etc?

 If sure, did you define NCS groups in refinement? TLS refinement? Try
 different refinement programs?

 How big is the molecule? Was it solved by MR or experimental phasing?

 You can try superimposing A/B on C/D and refinement with tight NCS then
 adjust NCS restraints during model adjustments based on local differences
 or also see if phenix autobuild helps.

 Best,
 Debanu
 --
 Debanu Das
 Accelero Biostructures


 On Jan 24, 2017, at 8:42 PM, Pooja Kesari  wrote:

 Dear All,

 I have a 2.6 A resolution structure having four chains in an asymmetric
 unit.
 The chain A and B have density for almost all residues however we don't
 have proper residue density in chain C and D.What can be tried to build
 chain C and D ?



 Many Thanks
 Pooja


>>>
>>>
>>> --
>>> Thanks & Regards,
>>> Pooja Kesari
>>> Research Scholar
>>> Department Of Biotechnology
>>> Indian Institute of Technology Roorkee
>>> INDIA
>>>
>>>
>>
>
>
> --
> Thanks & Regards,
> Pooja Kesari
> Research Scholar
> Department Of Biotechnology
> Indian Institute of Technology Roorkee
> INDIA
>
>

Re: [ccp4bb] Bad density for chains

[Pooja Kesari]

We have a 2.6 A structure showing four chains in an asymmetric unit. Our
protein is 360 residues around 40 kDa . Mattews shows four chain in an
assymetric unit (solvent 49% mattews coeff 2.44). The template has about
60% homologous with our protein. The molecular replacement against this
template gave an initial free R of 38.  We did chain tracing and found that
we have good density (2Fo-Fc) for chain A and B but poor density for C and
D.

1. The density for a particular stretch of 10 amino acids (disordered loop
region) is absent in all the chains. We could not found density for this
flexible loop region in any of the already known structures. Any suggestion
on how can we build this region?

2. We did not find density for most of the loop regions in chain C and D
which were well traced in chain A and B. How can we improving the density
for these two chains based on chain A and B (Density modification)?

3. We analysed the data using phenix xtriage and found that our data shows
severe anisotropy. Any suggestion of anisotropy correction?

Pointless and Ctruncate analyses didn't show twinning or NCS.  I have
checked the space group using Zanuda. We are stuck at a free value of 32.

On Thu, Jan 26, 2017 at 4:47 PM, Eleanor Dodson 
wrote:

> This is a bit too vague to help much.
> How did you solve the structure?
> Eleanor
>
> On 26 January 2017 at 03:50, Pooja Kesari  wrote:
>
>> Dear All,
>> Thank you all for reply.
>>
>> We have checked the data for twinning.
>> Our protein is 360 residues around 40 kDa protein.
>> We have tried TLS refinement.
>> chain A and B don't superimpose well with chain C and D. (A and B chains
>> also share slight difference )
>> Since we don't have proper density for *some regions*  chain C and D, we
>> are not sure whether these chain have similar or different conformations.
>> We tried anisotropy correction and the model refined a bit.
>>
>>
>> On Wed, Jan 25, 2017 at 10:32 AM, Debanu  wrote:
>>
>>> Hi Pooja,
>>>
>>> Are you positive you have the correct space group and there are no other
>>> issues like twinning, etc?
>>>
>>> If sure, did you define NCS groups in refinement? TLS refinement? Try
>>> different refinement programs?
>>>
>>> How big is the molecule? Was it solved by MR or experimental phasing?
>>>
>>> You can try superimposing A/B on C/D and refinement with tight NCS then
>>> adjust NCS restraints during model adjustments based on local differences
>>> or also see if phenix autobuild helps.
>>>
>>> Best,
>>> Debanu
>>> --
>>> Debanu Das
>>> Accelero Biostructures
>>>
>>>
>>> On Jan 24, 2017, at 8:42 PM, Pooja Kesari  wrote:
>>>
>>> Dear All,
>>>
>>> I have a 2.6 A resolution structure having four chains in an asymmetric
>>> unit.
>>> The chain A and B have density for almost all residues however we don't
>>> have proper residue density in chain C and D.What can be tried to build
>>> chain C and D ?
>>>
>>>
>>>
>>> Many Thanks
>>> Pooja
>>>
>>>
>>
>>
>> --
>> Thanks & Regards,
>> Pooja Kesari
>> Research Scholar
>> Department Of Biotechnology
>> Indian Institute of Technology Roorkee
>> INDIA
>>
>>
>


-- 
Thanks & Regards,
Pooja Kesari
Research Scholar
Department Of Biotechnology
Indian Institute of Technology Roorkee
INDIA

Re: [ccp4bb] Bad density for chains

[Eleanor Dodson]

This is a bit too vague to help much.
How did you solve the structure?
Eleanor

On 26 January 2017 at 03:50, Pooja Kesari  wrote:

> Dear All,
> Thank you all for reply.
>
> We have checked the data for twinning.
> Our protein is 360 residues around 40 kDa protein.
> We have tried TLS refinement.
> chain A and B don't superimpose well with chain C and D. (A and B chains
> also share slight difference )
> Since we don't have proper density for *some regions*  chain C and D, we
> are not sure whether these chain have similar or different conformations.
> We tried anisotropy correction and the model refined a bit.
>
>
> On Wed, Jan 25, 2017 at 10:32 AM, Debanu  wrote:
>
>> Hi Pooja,
>>
>> Are you positive you have the correct space group and there are no other
>> issues like twinning, etc?
>>
>> If sure, did you define NCS groups in refinement? TLS refinement? Try
>> different refinement programs?
>>
>> How big is the molecule? Was it solved by MR or experimental phasing?
>>
>> You can try superimposing A/B on C/D and refinement with tight NCS then
>> adjust NCS restraints during model adjustments based on local differences
>> or also see if phenix autobuild helps.
>>
>> Best,
>> Debanu
>> --
>> Debanu Das
>> Accelero Biostructures
>>
>>
>> On Jan 24, 2017, at 8:42 PM, Pooja Kesari  wrote:
>>
>> Dear All,
>>
>> I have a 2.6 A resolution structure having four chains in an asymmetric
>> unit.
>> The chain A and B have density for almost all residues however we don't
>> have proper residue density in chain C and D.What can be tried to build
>> chain C and D ?
>>
>>
>>
>> Many Thanks
>> Pooja
>>
>>
>
>
> --
> Thanks & Regards,
> Pooja Kesari
> Research Scholar
> Department Of Biotechnology
> Indian Institute of Technology Roorkee
> INDIA
>
>