Re: [ccp4bb] low resolution data refinement

[M T]

Dear Liu,

If you are really confident in your MR solution some good tools to refine low 
resolution structures are LORESTR pipeline and iSOLDE software.

Best.

Michel.

> Le 21 nov. 2023 à 13:03, Yahui Liu  a écrit :
> 
> 
> Dear all,
> I got a protein crystal dataset of 4.3 A and would like to some the structure 
> with MR.
> Now I am suffering with the refinement. I used both Refmac and Phenix.
> 
> Someone could give me a hand or  any suggestions?
> 
> All the best
> 
> To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] What could these crystals be?

[M T]

Dear Careina,

If you have easy access to mass spectrometry, you can try to fish/rince your « 
pumpkin seeds » and send them to mass to try to identify what is inside to see 
if it needs optimization or not.

Best.

> Le 8 nov. 2023 à 16:03, 02531c126adf-dmarc-requ...@jiscmail.ac.uk a écrit 
> :
> 
> 
> Hi all
> We have been trying with no success to crystalize a protein. Recently we got 
> these strange shape "crystals". They are hard and flat but they do not 
> diffract at all. Any ideas as to what could cause this?
> Careina
> 
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> 
> 



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[ccp4bb] Overrefinement considerations and Refmac5.

[M T]

Dear BBers,

I am trying to refine a structure using COOT and Refmac5 and I have some
concerns about overrefinement and x-ray term weight in Refmac5, based on
the fact that during refinement to let R factor to drift too far from Rfree
is not good...

So... First question about that : what is too far ? I have some values in
mind like 6% of difference is OK, 10% is not... But is there a relation in
between resolution of the structure and this difference? Should it be
higher at lower resolution, or always around 6-7% independently of the
resolution?

Second question is, ok, I have a too big difference, lets say 9-10%... What
could be the reason of that and on what to play to reduce this difference?

One way I choose is to look at the x-ray term weight (even if I am totally
sure that Refmac5 is doing things better than me), because I saw that the
final rms on BondLength were to constraint (I have in mind that this value
should stays in between 0.02 and 0.01).
So I looked into Refmac log to know where was the starting point and I
found 8.75.
Then I tried several tests  and here are the results:
*

R factor

Rfree
BondLength

BondAngle

ChirVolume

Auto weighting and experimental sigmas boxes checked

0.1932
0.2886

0.0072

1.6426

0.1184

Weighting term at 4 and experimental sigmas box checked

0.1780
0.3159

0.1047

8.1929

0.5937

Weighting term at 4

0.1792
0.3143

0.1008

7.8200

0.5667

Weighting term at 15 and experimental sigmas box checked

0.1783
0.3272

0.2020

1.6569

0.9745

Weighting term at 15

0.1801
0.3279

0.2022

12.5748

0.9792

Weighting term at 8.75

0.1790
0.3235

0.1545

10.5118

0.7909

Auto weighting box checked

0.1948
0.2880

0.0076

1.6308

0.1176



*Refinement Parameters*
[image: image.png]

So like nothing looks satisfying I decided to ask my questions here...

What do you recommend to fix my problem, which is a too large difference
between R and Rfree?

Thank you for answers.



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Re: [ccp4bb] Refmac: problem with anisotropic refinement of heavy atoms.

[M T]

Sorry, I moved the image before the sending of the mail and then I was not
attached.

2018-04-04 11:26 GMT+02:00 M T <michel...@gmail.com>:

> Hello,
>
> I am refining a structure at 2.1Å, using Refmac and manual constructions
> in coot.
> I have few Ca, Cl and Mg in my structure and some of them have a clearly
> anisotropic distribution, then I decided to use ANISOU line in pdb file
> only for these atoms.
> In refinement parameters of Refmac I set "mixed (isotropic/anisotropic)
> temperature factors".
> And I have a problem with some of them which are obviously badly refined
> (see attached picture).
>
> What could be the reason of that? What should I badly set?
>
> Thank you for your help.
>

[ccp4bb] Refmac: problem with anisotropic refinement of heavy atoms.

[M T]

Hello,

I am refining a structure at 2.1Å, using Refmac and manual constructions in
coot.
I have few Ca, Cl and Mg in my structure and some of them have a clearly
anisotropic distribution, then I decided to use ANISOU line in pdb file
only for these atoms.
In refinement parameters of Refmac I set "mixed (isotropic/anisotropic)
temperature factors".
And I have a problem with some of them which are obviously badly refined
(see attached picture).

What could be the reason of that? What should I badly set?

Thank you for your help.

Re: [ccp4bb] coot: obtaining a clickable list of outiers in a Ramachandran plot

[M T]

In coot you have also the possibility to zoom into the ramachandran, this can 
helps maybe.

Michel.

> Le 22 mars 2018 à 18:57, Ashley Pike  a écrit :
> 
> Running phenix.molprobity yourpdbfile from command-line used to work – will 
> give a molprobity_coot.py (from which when you calculate>run script in coot 
> you will get a clickable list of rama/rotamer outliers and clashes)
>  
> ..although just checking it appears to not work with current COOT version 
> distributed with CCP4 :(
>  
> Does appear to work om my linux box if you launch coot via phenix (from 
> command link type phenix.start_coot)
>  
> HTH,
> Ash
>  
> From: CCP4 bulletin board  On Behalf Of Kabasakal, 
> Burak V
> Sent: 22 March 2018 11:04
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] coot: obtaining a clickable list of outiers in a 
> Ramachandran plot
>  
> You can evaluate your structure in MolProbity, then it would give a list of 
> Ramachandran outliers similar to the output of COOT.
>  
> http://molprobity.biochem.duke.edu/index.php?MolProbSID=ktiuaj4c1v48mlgqq3inopfrf6=22
>  
> Regards,
>  
> Burak 
> From: CCP4 bulletin board  on behalf of Robbie Joosten 
> 
> Sent: 22 March 2018 10:52:57
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] coot: obtaining a clickable list of outiers in a 
> Ramachandran plot
>  
> Dear Laurant,
> 
> Perhaps not a complete answer to your question, but more of a strategy. You 
> can let the Ramachandran plot in COOT only show the outliers which would make 
> everything a bit more tangible. Then only go for the massive outliers, these 
> are the problems that need rebuilding (e.g. through peptide flipping). Many 
> of the marginal outliers can be fixed through reciprocal space refinement 
> with proper restraint weighting. Shameless plug: try PDB-REDO. It is (most of 
> the time) quite good at tidying up your Ramachandran plot.
> 
> Cheers,
> Robbie
> 
> > -Original Message-
> > From: CCP4 bulletin board  On Behalf Of
> > maveyrau
> > Sent: Thursday, March 22, 2018 11:27
> > To: CCP4BB@JISCMAIL.AC.UK
> > Subject: [ccp4bb] coot: obtaining a clickable list of outiers in a 
> > Ramachandran
> > plot
> > 
> > Hi all,
> > 
> > I’m currently refining quite a big oligomer (about 3500 residues) using
> > refmac/coot, at a 2.85 A resolution. The Ramachandran tool in Coot indicates
> > about 100 outliers, and I would like to check them individually… Is there a
> > way to get a clickable list of outliers? I know I can click on the 
> > Ramachandran
> > plot directly, but with so many residues it’s quite impossible to go through
> > all of them…
> > 
> > Thanks for any advices…
> > Laurent
> > --
> > Laurent Maveyraud laurent.maveyraud AT ipbs DOT fr
> > P I C T   ---  Plateforme Intégrée de Criblage de Toulouse
> > Université  Paul  Sabatier /  CNRS  /  I.P.B.S.  UMR  5089
> > Département BiologieStructurale   et   Biophysique
> > http://cribligand.ipbs.fr   http://www.ipbs.fr
> > 205 route de Narbonne  31077 TOULOUSE Cedex FRANCE
> > Tél: +33 (0)561 175 435   Mob.: +33 (0)646 042 111
> > --
> > 
> > 
> >

Re: [ccp4bb] AW: [ccp4bb] Small molecule not refined in coot.

[M T]

So finally the solution was easy access...

Thanks to someone who suggest me to do that, I created a new user, to
verify if coot was ok under that session, and it was fine.
Then I went back on my session, I removed the files below:
0-coot-history.py
0-coot-history.scm
0-coot.state.py
0-coot.state.scm

I restarted Coot, and loaded my files (.pdb, .mtz and .cif), Coot didn't
crash and I am now able to do a real space refinement.

It should be my first intention to remove Coot preferences.

Regards.

2018-03-12 21:39 GMT+01:00 Paul Emsley <pems...@mrc-lmb.cam.ac.uk>:

>
> On 12/03/18 14:05, M T wrote:
> > Dear all,
> >
> > I restart this topic because the problem was finally not solved...
>
> You give me an opportunity to comment, I had previously missed the boat
> while traveling.
>
> >
> > Summary:
> > - I am working on a structure with an unnatural ligand and I want to
> > refine this ligand using Coot.
>
> Sounds like a fine plan.
>
> > - Each time I try to import the .cif of my ligand produced by PRODRG
> > web server or through CCP4/ProDrg, coot crashes with error message
> > below (see quoted messages).
>
> The first thing to do, when Coot crashes is to check for a new revision.
> There's a good chance that the problem has been fixed and is just
> waiting for you to download it.
>
> > - I sent my files to someone else and they are working on his computer
> > (both are Mac and are using CCP4/Coot on same SBGRID server, with same
> > files, and macOS was different).
> > - I saw that my macOS was outdated (Starting SBGRID saying "- MacOS X
> > versions 10.10 (Yosemite) and earlier are no longer officially
> > supported").
> > - I did an update of my macOS to High Sierra (10.13.3).
> > - I retried with same files, Coot crashed.
>
> For the record, the crash log would be highly helpful.
>
> > - I went back to PRODRG server to generate a simple .cif
> > (CH3-CH2-CH2-CH3).
>
> Fine as it was in its day, PRODRG is no longer what we use for ligands.
> We use Acedrg.  If you want to use SMILES, you can use it on the command
> line (that's what I do). If you want to use it from a sketch, use the
> Ligand Builder in Coot. You can also use pyrogen (I do).
>
> > - I started Coot, opened the .cif file using "Import CIF
> > dictionary...", Coot crashed.
> >
> > It seems that opening any .cif file on my computer causes Coot crash.
>
> Edit -> Preferences -> File Selector -> Modern File Chooser
>
> For the record (again) this is the site that I use to download Coot mac
> binaries:
>
> http://scottlab.ucsc.edu/xtal/wiki/index.php/Stand-Alone_Coot
>
> >
> >
> > Anybody has an idea to solve the problem?
>
> It was discussed on the mailing list in January and fixed by the next day.
>
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=ind1801=COOT===5852
>
> Regards,
>
> Paul.
>
>

Re: [ccp4bb] AW: [ccp4bb] Small molecule not refined in coot.

[M T]

Dear all,

I restart this topic because the problem was finally not solved...

Summary:
- I am working on a structure with an unnatural ligand and I want to refine
this ligand using coot.
- Each time I try to import the .cif of my ligand produced by PRODRG web
server or through CCP4/ProDrg, coot crashes with error message below (see
quoted messages).
- I sent my files to someone else and they are working on his computer
(both are Mac and are using CCP4/Coot on same SBGRID server, with same
files, and macOS was different).
- I saw that my macOS was outdated (Starting SBGRID saying "- MacOS X
versions 10.10 (Yosemite) and earlier are no longer officially supported").
- I did an update of my macOS to High Sierra (10.13.3).
- I retried with same files, Coot crashed.
- I went back to PRODRG server to generate a simple .cif (CH3-CH2-CH2-CH3).
- I started Coot, opened the .cif file using "Import CIF dictionary...",
Coot crashed.

It seems that opening any .cif file on my computer causes Coot crash.

Here are the terminal lines for Coot starting, .cif opening and crash; in
which there is some WARNING I can't really understand completely (I'm not
an unix power user).

INFO:: Using Standard CCP4 Refmac dictionary from CLIBD_MON:
> /programs/i386-mac/ccp4/7.0/ccp4-7.0/lib/data/monomers/
> INFO:: Reading coordinate file:
> /programs/i386-mac/ccp4/7.0/ccp4-7.0/share/coot/standard-residues.pdb
>  PDB file
> /programs/i386-mac/ccp4/7.0/ccp4-7.0/share/coot/standard-residues.pdb has
> been read.
> Spacegroup: P 1
> initalize graphics molecules...done.
> (filter-fileselection-filenames-state)
> (get-active-map-drag-flag)
> (use-graphics-interface-state)
> INFO:: coot.py imported
>
> ** (coot-bin:2710): WARNING **: Trying to register gtype
> 'GMountMountFlags' as enum when in fact it is of type 'GFlags'
>
> ** (coot-bin:2710): WARNING **: Trying to register gtype
> 'GDriveStartFlags' as enum when in fact it is of type 'GFlags'
>
> ** (coot-bin:2710): WARNING **: Trying to register gtype 'GSocketMsgFlags'
> as enum when in fact it is of type 'GFlags'
> INFO:: coot_python initialized
> Running python script
> /programs/i386-mac/ccp4/7.0/ccp4-7.0/lib/python2.7/site-packages/coot/coot_load_modules.py
> Good Afternoon Username, Welcome to Coot version 0.8.9
> (set-display-intro-string "Good Afternoon Username, Welcome to Coot
> version 0.8.9")
> Coot Python Scripting GUI code found and loaded.
> Coot Python Scripting GUI code found and loaded.
> WARNING:: no directory "/programs/share/coot/pyextras" in
> COOT_PYTHON_EXTRAS_DIR
> /programs/share/coot/pyextras:/Users/Username/.coot-pyextras
> WARNING:: no directory "/Users/Username/.coot-pyextras" in
> COOT_PYTHON_EXTRAS_DIR
> /programs/share/coot/pyextras:/Users/Username/.coot-pyextras
> (use-graphics-interface-state)
> (filter-fileselection-filenames-state)
> (get-active-map-drag-flag)
> (use-graphics-interface-state)
> Coot Scheme Scripting GUI code found and loaded.
> Good afternoon Username. Welcome to Coot 0.8.9.
> (set-display-intro-string "Good afternoon Username. Welcome to Coot 0.8.9")
> (set-display-lists-for-maps 0)
> load /Users/Username/.coot-preferences/coot-preferences.scm
> (set-filter-fileselection-filenames 0)
> (unset-sticky-sort-by-date)
> (set-colour-map-rotation-on-read-pdb 20.30)
> (set-colour-map-rotation-on-read-pdb-c-only-flag 1)
> (set-density-size 10.00)
> (set-swap-difference-map-colours 0)
> (set-active-map-drag-flag 1)
> (set-idle-function-rotate-angle  1.00)
> (filter-fileselection-filenames-state)
> (get-active-map-drag-flag)
> (use-graphics-interface-state)
> (first-coords-imol)
>
> ** (coot-bin:2710): WARNING **: Widget not found:
> cif_dictionary_file_selector_create_molecule_checkbutton
> /programs/i386-mac/ccp4/7.0/ccp4-7.0/bin/coot: line 288:  2710
> Segmentation fault: 11  $coot_bin "$@"
> catching the crash log:
> coot-exe: "/programs/i386-mac/ccp4/7.0/ccp4-7.0/libexec/coot-bin"
> coot-version:
> /programs/i386-mac/ccp4/7.0/ccp4-7.0/libexec/coot-bin
> platform:
> /usr/bin/uname
> core: #f
> No core file found.  No debugging
>


Anybody has an idea to solve the problem?

Thanks.

Michel.

2018-03-06 16:03 GMT+01:00 M T <michel...@gmail.com>:

> Last update...
>
> Everything works well with the same files and the same Coot version on an
> other computer. The problem may come from the os of my computer which is
> too old and has to be upgraded.
>
> Thank you for your help.
>
> 2018-03-06 9:20 GMT+01:00 M T <michel...@gmail.com>:
>
>> Dear all,
>>
>> I did something very simple...
>>
>> Starting from the pdb I obtained from PRODRG server (manual drawing, then
>> transfer to P

Re: [ccp4bb] AW: [ccp4bb] Small molecule not refined in coot.

[M T]

Last update...

Everything works well with the same files and the same Coot version on an
other computer. The problem may come from the os of my computer which is
too old and has to be upgraded.

Thank you for your help.

2018-03-06 9:20 GMT+01:00 M T <michel...@gmail.com>:

> Dear all,
>
> I did something very simple...
>
> Starting from the pdb I obtained from PRODRG server (manual drawing, then
> transfer to PRODRG and download of the pdb from server), in CCP4 I used
> ProDrg to generate a new .cif and a new .pdb.
> After I used "View Job Results (ew style)" to open my new pdb in coot, I
> loaded the .mtz coming from the previous run of Refmac (in which I have a
> nice green blob corresponding to my ligand), I tried "Real Space Refine
> Zone" and ofc "Failed to find restraints for: DRG" since no .cif is loaded.
> I tried to import the .cif coming from CCP4/ProDrg, using "Import CIF
> dictionary..." and Coot crashed with this message:
>
> ** (coot-bin:5539): WARNING **: Widget not found:
>> cif_dictionary_file_selector_create_molecule_checkbutton
>> /programs/i386-mac/ccp4/7.0/ccp4-7.0/bin/coot: line 288:  5539
>> Segmentation fault: 11  $coot_bin "$@"
>> catching the crash log:
>> coot-exe: "/programs/i386-mac/ccp4/7.0/ccp4-7.0/libexec/coot-bin"
>> coot-version:
>> /programs/i386-mac/ccp4/7.0/ccp4-7.0/libexec/coot-bin
>> platform:
>> /usr/bin/uname
>> core: #f
>> No core file found.  No debugging
>>
>
> At the loading of the molecule I have these messages:
>
> INFO:: Reading coordinate file: the path to my .pdb
>>  PDB file my .pdb has been read.
>> No Spacegroup found for this PDB file
>> INFO:: Found 1 models
>>Model 1 had 0 links
>> WARNING:: No symmetry available for this molecule
>> WARNING:: No Symmetry for this model
>> in add_molecular_symmetry_matrices() made 0 biomt matrices
>> Molecule 0 read successfully
>> DEBUG:: there were 1 types with no dictionary
>> INFO:: comp-id: DRG is marked for non-autoloading - stopping now
>> (no-coot-tips)
>> (use-graphics-interface-state)
>>
>
> And nothing special at the reading of the .mtz file.
>
> The comparison of the naming in .pdb and .cif file reveals no evident
> difference (same molecule name DRG, and same atom names... I just compare
> the first 10/15 atom names).
> Is it a problem coming from my ligand, from Coot?...
> Could it be something related to that:
>
>> # WARNING: REFMAC5 uses columns 77-78 of PDB ATOM records to
>> #  establish equivalence between model and topology. If
>> #  you use O or other programmes that produce defective
>> #  PDB files you must restore these columns, otherwise
>> #  REFMAC5 will not recognise this topology.
>>
>  Atom names are at different columns if the file is coming from
> CCP4/ProDrg or from Coot. Columns 77-78 in the file saved from Coot and
> columns 79-80 if the file is generated from CCP4/ProDrg.
>
> Thank you for your help.
>
>
> 2018-03-05 16:57 GMT+01:00 Eleanor Dodson <176a9d5ebad7-dmarc-
> requ...@jiscmail.ac.uk>:
>
>> Herman is right - as long as refmac reads your generated DRG that is the
>> dictionary it will use..
>>
>> My way of doing this sort of thing:
>>
>> Start with COOT with the DRG coordinates and ProDRG dictionary and try a
>> bit of real space refinement.
>> Coot should move the coodinates a bit and you should see something happen.
>>
>> Then read out those new coordinates and run refmac. Again the coords
>> should move a bit, and at lest the B factors should change. If REFMAC
>> dooesnt like your naming it will report it..
>>
>> Again if COOT accepted the dictionary the first time I cant see why it
>> wont read it again. are you sure you are finding the same cif file?
>>
>> You will have to read your own DRG into coot or otherwise it will find
>> something which is not appropriate for your problem..
>> Eleeanor
>>
>> On 5 March 2018 at 13:36, <herman.schreu...@sanofi.com> wrote:
>>
>>> PS: If you have two different home-brewn ligands, you have to rename one
>>> of them (pdb and cif), otherwise the same dictionary will be applied to two
>>> different ligands. Also make sure your cif file is a dictionary and not
>>> just a coordinate file.
>>>
>>> HS
>>>
>>>
>>>
>>> *Von:* Schreuder, Herman /DE
>>> *Gesendet:* Montag, 5. März 2018 14:31
>>> *An:* 'Colin Levy'; CCP4BB@JISCMAIL.AC.UK
>>> *Betreff:* AW: [ccp4bb] Small molecule not refi

Re: [ccp4bb] AW: [ccp4bb] Small molecule not refined in coot.

[M T]

 atom
>> names of the pdb file do match the residue and atom names in the cif
>> dictionary.
>>
>>
>>
>> Best,
>>
>> Herman
>>
>>
>>
>> *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK
>> <CCP4BB@JISCMAIL.AC.UK>] *Im Auftrag von *Colin Levy
>> *Gesendet:* Montag, 5. März 2018 13:38
>> *An:* CCP4BB@JISCMAIL.AC.UK
>> *Betreff:* [EXTERNAL] Re: [ccp4bb] Small molecule not refined in coot.
>>
>>
>>
>> Hi Michel,
>>
>>
>>
>> If your ligand is designated as DRG in your pdb then refinement programs
>> will anticipate that it is:
>>
>>
>> Chemical Description
>>
>> *Name*
>>
>> 5,6-DIHYDRO-BENZO[H]CINNOLIN-3-YLAMINE
>>
>> *Formula*
>>
>> C12 H11 N3
>>
>> *Formal charge*
>>
>> 0
>>
>> *Molecular weight*
>>
>> 197.236 g/mol
>>
>> *Component type*
>>
>> NON-POLYMER
>>
>>
>>
>>
>>
>> If this is not the case then you need to choose a unique identifier, this
>> should then allow your cif library to be utilised appropriately during
>> refinement.
>>
>>
>>
>> Colin
>>
>>
>>
>>
>>
>>
>>
>> Dr. Colin W. Levy
>>
>> MIB G016
>>
>> Tel.  0161 275 5090
>>
>> Mob.07786 197 554
>> c.l...@manchester.ac.uk
>>
>>
>>
>>  Manchester Institute of Biotechnology | University of Manchester | 3.020
>> Garside Building | 131 Princess Street | Manchester | M1 7DN
>> <https://maps.google.com/?q=131+Princess+Street+%7C+Manchester+%7C+M1+7DN=gmail=g>
>>
>>
>>
>> <https://urldefense.proofpoint.com/v2/url?u=https-3A__www.facebook.com_MIB2006_-3Fref-3Daymt-5Fhomepage-5Fpanel=DwMFAg=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ=2Ib8W-dQ0MHoUt4AHNaero85sr0-ZRZXc93QBAWf108=bKUGrH6wK5dQ2KLRw6oxFvnOjjWsZxfqbmZabgHnlnE=>
>> <https://urldefense.proofpoint.com/v2/url?u=https-3A__plus.google.com_u_0_b_111834792186886067949_111834792186886067949_posts-3F-5Fga-3D1.142941878.209859079.1446729440=DwMFAg=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ=2Ib8W-dQ0MHoUt4AHNaero85sr0-ZRZXc93QBAWf108=MxPD1QWp-GEhbvwGRdaEozWff1HvapFjYoa0O8j8ARY=>
>> <https://urldefense.proofpoint.com/v2/url?u=https-3A__www.linkedin.com_company_manchester-2Dinstitute-2Dof-2Dbiotechnology-3Ftrk-3Dbiz-2Dcompanies-2Dcym=DwMFAg=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ=2Ib8W-dQ0MHoUt4AHNaero85sr0-ZRZXc93QBAWf108=NX4ub_6zOoHnKdhhIKKDNhy_ubtCXCpBDqtdRB6ChmM=>
>> <https://urldefense.proofpoint.com/v2/url?u=https-3A__twitter.com_-3Flang-3Den-2Dgb-26lang-3Den-2Dgb=DwMFAg=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ=2Ib8W-dQ0MHoUt4AHNaero85sr0-ZRZXc93QBAWf108=gNH6h2r1l3Fb1ho0UL9pWdjiU7yWfDS8tojGhnLoQE4=>
>> <https://urldefense.proofpoint.com/v2/url?u=https-3A__www.youtube.com_channel_UCCBV8zDxk4L9yOEcujvSFNw=DwMFAg=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ=2Ib8W-dQ0MHoUt4AHNaero85sr0-ZRZXc93QBAWf108=z2OpzMpJB9EYcL5yPECYcsocmhXktsEK-N_415fT35g=>
>>
>>
>>
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>>
>> <https://urldefense.proofpoint.com/v2/url?u=https-3A__www.youtube.com_channel_UCCBV8zDxk4L9yOEcujvSFNw=DwMFAg=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ=2Ib8W-dQ0MHoUt4AHNaero85sr0-ZRZXc93QBAWf108=z2OpzMpJB9EYcL5yPECYcsocmhXktsEK-N_415fT35g=>
>>
>>
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>>
>>
>>
>> On 5 Mar 2018, at 12:30, M T <michel...@gmail.com> wrote:
>>
>>
>>
>> Dear all,
>>
>> I am actually dealing with a structure containing an unnatural ligand.
>>
>> I generated the pdb file by drawing it on PRODRG server, I did a manual
>> pre-fit in the map using coot and I manually merged the pdf file of my
>> protein with the one of the ligand.
>>
>> After that I did few cycles of refinement using Refmac5, with my mtz, my
>> merged pdb file and the cif library of the ligand as input files.
>>
>> First I saw that even if Refmac "completed succesfully" it didn't modify
>> coordinates of the ligand, second I saw a warning about the "DRG" molecule
>> in the log of Refmac (WARNING: duplicated name of monomer DRG Last entry
>> will be used.), third in Coot I cannot use "Real Space Refine Zone" with
>> the ligand and when I try to import the CIF dictionary produced by PRODRG
>> (through the server or even the one generated through CCP4 ProDrg), it
>> causes Coot crash (** (coot-bin:4467): WARNING **: Widget not found:
>> cif_dictionary_file_selector_create_molecule_checkbutton
>> /programs/i386-mac/ccp4/7.0/ccp4-7.0/bin/coot: line 288:  4467
>> Segmentation fault: 11  $coot_bin "$@").
>>
>> Clearly I am not a skilled user of all these programs, so I certainly did
>> a/some mistake/s, but if someone can give me tips to be able to refine my
>> ligand... Should I rename my DRG molecule, should I verify particular
>> things, should I use other ways to generate the pdb and the library of my
>> ligand?
>>
>> Thank you.
>>
>>
>>
>> P.S.: I am running CCP4 and coot using SBGRID on a Mac.
>>
>> Michel
>>
>>
>>
>>
>>
>
>

[ccp4bb] Small molecule not refined in coot.

[M T]

Dear all,

I am actually dealing with a structure containing an unnatural ligand.
I generated the pdb file by drawing it on PRODRG server, I did a manual
pre-fit in the map using coot and I manually merged the pdf file of my
protein with the one of the ligand.
After that I did few cycles of refinement using Refmac5, with my mtz, my
merged pdb file and the cif library of the ligand as input files.

First I saw that even if Refmac "completed succesfully" it didn't modify
coordinates of the ligand, second I saw a warning about the "DRG" molecule
in the log of Refmac (WARNING: duplicated name of monomer DRG Last entry
will be used.), third in Coot I cannot use "Real Space Refine Zone" with
the ligand and when I try to import the CIF dictionary produced by PRODRG
(through the server or even the one generated through CCP4 ProDrg), it
causes Coot crash (** (coot-bin:4467): WARNING **: Widget not found:
cif_dictionary_file_selector_create_molecule_checkbutton
/programs/i386-mac/ccp4/7.0/ccp4-7.0/bin/coot: line 288:  4467 Segmentation
fault: 11  $coot_bin "$@").

Clearly I am not a skilled user of all these programs, so I certainly did
a/some mistake/s, but if someone can give me tips to be able to refine my
ligand... Should I rename my DRG molecule, should I verify particular
things, should I use other ways to generate the pdb and the library of my
ligand?

Thank you.

P.S.: I am running CCP4 and coot using SBGRID on a Mac.

Michel

Re: [ccp4bb] Protein Ligand interaction using SPR technique

[M T]

Dear Praveen,

As said, Biacore3000 is not the best for small molecules, but in some case you 
can do nice things with this system. I see 3 possibilities :
- You can immobilize your molecules on the chip if you have an amine on it 
which is supposed to not participate to the interaction.
- You can have also the possibility to use carrier protein as BSA which is 
often found labeled with various small molecules and why not yours.
- You can also use competition assays if your small molecules are supposed to 
inhibit larger molecules interaction.

I am agree with the problems of non stable baseline with NiNTA chips, covalent 
interactions are better, but in these cases regeneration can be more 
difficult...

An other thing you can consider is streptavidin surfaces, especially if you can 
obtain your small molecules biotinylated. But in that case, long linkers should 
be used in case of your small molecules may be buried in an hydrophobic pocket.

If you cannot fit in these case you should consider other experiments as ITC, 
NMR (STD transfert)...

Good luck.

Michel.

> Le 31 mars 2017 à 07:01, Praveen Tripathi  a 
> écrit :
> 
> Dear all,
> Sorry for off-topic question.
> I want to study protein interaction with few small molecules using SPR 
> (Machine- BIACORE 3000).
> 
> The recombinant protein expressed in bacterial expression system is of 92 kDa 
> (with His tag), pI= 9.
> 
> Question- 
> 1. What should be the chip of choice- NTA chip or CM5 chip of GE healthcare?
> 2. Is there any alternative available for chips and machine?
> 
> Thanks in advance.
> 
> Regards
> 
> Praveen

Re: [ccp4bb] HETATM automated chain assignment

[M T]

Dear Romain,

I already ask this question to someone of the pdb staff during a deposition
process, and he answer me that it is an in house program and they don't
distribute theirs in house programs, so if this direction hit your mind,
you can forget it directly.

Meow...

2013/2/15 Talon Romain talon@gmail.com

 Hello to the CCP4 bulletin board community,

 I would like to know if I could find a tool to automatically assign HETATM
 atom (or even, water molecules) to the nearest protein chain ?

 In my case, I have 4 protein chains in the asymmetric unit : A, B, C and
 D. I would like to assign each ions and each ligands (which are numerous)
 with the chain letter of the nearest residue that coordinate them.
 Usually, I rename everything by hand but as the in-house program of the
 PDBe AutoDep deposition tool automatically do that...

 I beg your pardon if this question has just been posted here. I didn't
 find any tool either in the CCP4 Suite or in the Extensions and
 Calculate menus of the Coot program (v0.7).

 Best regards.

 Romain Talon

Re: [ccp4bb] MR - small coiled coil, 1.65A = 1.000 solutions, all of them wrong

[M T]

Dear Napoleão, we solved this type of problem in a paper of 2004 (J. Mol.
Biol. (2004) 342, 275–287) with the using of EPMR which use a evolutionary
search algorithm, I will send you the paper later. It was 36aa dimeric
coiled-coil and we had a lot of molecular replacement problems with other
tested molecular replacement programs.

http://www.epmr.info/UsersGuide.html

Good luck.

Re: [ccp4bb] Concentrating a protein solution - subbu

[M T]

A solution could be to control the solubility of your protein in different
pH and salts (you can also add some additives) by using the Thermal Shift
Assay. You may find a better buffer in which to concentrate your protein.

If you think that the reason of the low diffraction is the quality of your
crystals try the suggestion of Eric, but if you think that is due to the
small size of the crystals, you can also try to make drops of 3µl (2µl of
protein and 1µl of the well) or to feed your crystals during the growing
(i.e. adding 0.5µl of protein solution to your drop after when you see that
the crystals stop to grow), or to do seeding like Eric suggests, but in big
drops (5µl in hanging or more in sitting drop) and with serial dilutions of
the seeds.

Good luck...

Michel.

Dear All:
 We have been trying to crystallize a protein which is large -  100 kDa.
 This is soluble but the best we can get is about 1 mg/mL.
 It did crystallize but did not diffract well. Efforts to increase the
 concentration has been unsuccessful. I am wondering whether there are
 methods that others use to increase the concentration other that using
 amicon columns.
 Any help will be appreciated.
 Thanks
 Subbu

Re: [ccp4bb] selenomethinine-labeled protein

[M T]

If you are afraid by the possible low incorporation of selenium in your
protein, and if you have some cysteines into your sequence, you can try the
double labeling -- Structure, Vol. 11, 1359–1367, November, 2003.

It works at the first try for me and I had no change in crystallization
conditions.

Michel.

2011/1/6 Yibin Lin yyb...@gmail.com

 Dear all,
 I try to express selenomethinine-labeled protein in E.coli methinoine
 auxotrophic strain B834 in M9 with 17 amino acid and 50 mg/ml se-met for 18
 hours at 37 degree. I don't why the colour of medium vary from colorless to
 yellow.  Could anybody tell me it is normal or not?

 Thanks a lot!

 Lin

Re: [ccp4bb] Cys auxotroph

[M T]

I think you should look at pubmed id 14604526, it works well for me. You should 
obtain the cells if you contact the authors. 

Michel.

Le 18 oct. 2010 à 17:27, Daniel Bonsor bon...@bbri.org a écrit :

 Have you looked at the Keio collection. Though they are not compatible with 
 T7 promoters, you can use the λDE3 Lysogenization Kit to add the T7 
 polymerase. 
 
 A possible source is from Yale http://cgsc.biology.yale.edu/Auxotrophs.php 
 though of course there are others.
 
 
 Dan

[ccp4bb] Alternate configurations blow-up in refmac

[M T]

Dear all,

We recently jump from a local version of ccp4 using Refmac_5.2.0019 to
an up-to-date version of ccp4 distributed by sbgrid using
Refmac_5.2.0102. From then, refinements with alternate configurations
are blowing-up in refmac. A similar problem was solved thanks to
ccp4bb ('Refmac dictionary problem' from  Simon Kolstoe on Fri, 17 Apr
2009  http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg10444.html
) by upgrading refmac..In our case, the behaviour of refmac
(blowing-up of the structure or not) with identical input files is
different when using the same version of the distributed program with
similar computers but with different environnements, which suggests
that the differences in behaviour should be due to local files. When
using the old version again, the alternate configurations now blow-up,
which means that some local files were modified... Which ones??? Any
idea for fixing the problem?


Thanks,

Michel

Re: [ccp4bb] Protein crystallizes while concentration

[M T]

I see some solutions to your problem and one works well for me on a small
protein domain.

I had exactly the same problem of crystallization during concentration and
in my case I solve the problem by heating the crystal suspension. The
validation of the protocol was made with support of 1D NMR to verify the
stability of the structure after heating.

So the solutions I see are:
- Concentrate your sample until apparition of crystals and mildly heat it
until solubilization of your crystals. Make a drop of 5µL in a
crystallization plate and let it cool down against 500µL of your buffer.
- Concentrate your sample until apparition of crystals and add the necessary
amount of water to solubilize your crystals. Make drops of 1 or 2µL in a
crystallization plate and let it concentrate against 500µL of your buffer.
- You can also choose the best solution (heating or dilution) to solubilize
your crystals ans try mild evaporation under paraffin oil to try to obtain
crystals.

And after, with the best solution, you can play with the salt concentration
of reservoir solution to try to obtain better crystals.

Gook luck.

Michel.


2010/2/18 Daniel Ryan d.z.r...@dundee.ac.uk

 A cheaper solution than buying the dialysis buttons would be just to make
 your own out of the top of a microtube. Depending on what volume you want to
 dialyse you can use either a PCR tube (~35 uL per lid) or larger 1.5 mL tube
 (~250 uL). Just cut the top off the tube using a hot scalpel, you then place
 your sample in the cap of the microtube, cover it with dialysis membrane and
 then secure the tubing using the top part of the tube that you cut-off.

 -Original Message-
 From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Lari
 Lehtiö
 Sent: 18 February 2010 16:38
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] Protein crystallizes while concentration

 Dear Rajkumar,

 I would use dialysis buttons.
 E.g. http://hamptonresearch.com/product_detail.aspx?cid=10sid=63pid=111

 Put your protein to the button and seal it with a piece of dialysis
 membrane. Place this to a linbro plate (easy to look with a
 microscope), fill the well with low salt buffer and seal with a cover
 slip.

 To make the diffusion slower, you can put the dialysis button inside a
 dialysis tubing with some high salt buffer and place this to a bigger
 volume of low salt buffer.


 ~L~

 __
 Lari Lehtiö
 Pharmacy, Department of Biochemistry and Pharmacy
 Åbo Akademi University,
 BioCity, FIN-20520 Turku
 Finland
 +358 2 215 4270
 http://www.users.abo.fi/llehtio/
 __


 Quoting E rajakumar e_rajaku...@yahoo.com:

  Dear All
  I am Rajkumar, working on the protein which has unusual behavior
  while concentration.
 
  When I kept protein in 15 mM Tris 7.5, 150 mM NaCl and 5 mM DTT, the
   solubility of the protein is decreases drastically and tend to
  crystallize while concentration.
  Protein cannot be concentrated more than 3 mg/mL, however I noticed
  white turbid protein if I force to concentrate 3mg/mL. When I
  observed this white turbid solution under the microscope, I noticed
  shower of tiny protein crystals which are needle in shape.
  I screened freshly purified protein (2.5 mg/mL) in different Hampton
   and Qiagen screens, strangely none of the conditions gave the
  crystals.  I concentrated left over protein at 15oC at 3 mg/mL and
  kept in the 4oC for 4 days again I noticed shower of crystals.
  This protein solubility is increased to ~20mg/mL when I kept in 15
  Tris 7.5, 400 mM NaCl, 5% Glycerol and 5 mM DTT, but did not
  crystallize while concentration and also after screening with
  Hampton and Qiagen screens.
 
  My queries are
  1. How do I get the crystals in the crystallization set up rather
  than while concentration, so that I can control the diffusion and
  finally nucleation?
  2. Could anybody give me suggestions on seeding in this type of
 situation?
  3. Any comments on reverse vapor diffusion for this type of protein
  are most welcome. So I can keep protein in high ionic strength (~400
   mM NaCl)and diffuse against low Ionic strength or deionized water?
  Or any other protocol?
  Any suggestions are well appreciated.
  Thanking you in advance
  Raj
 
  E. Rajakumara
  Postdoctoral Fellow  Strcutural Biology Program
  Memorial Sloan-Kettering Cancer Center
  New York-10021
  001 212 639 7986 (Lab)  001 917 674 6266 (Mobile)
 
 
Get your new Email address!
  Grab the Email name you#39;ve always wanted before someone else does!
  http://mail.promotions.yahoo.com/newdomains/aa/
 
 

Re: [ccp4bb] Hi prep column from GE

[M T]

What are the buffer pHs (first and second run), what is the
isoelectric point of your protein, have you tried Q column?... You
have to give more experimental details to be more precisely helped.

Nevertheless, you can find helpful advices in GE healthcare life
science Handbooks:
http://www4.gelifesciences.com/aptrix/upp01077.nsf/Content/orderonline_handbooks
Particularly in Protein Purification, Handbook and in Ion Exchange
Chromatography: Principles and methods.

Good luck Mike.

Re: [ccp4bb] Quick-soak

[M T]

2008/9/25 amit sharma [EMAIL PROTECTED]

 Dear CCP4bbers,
 I have a protein molecule(~9.0 kDa) that crystallized in the presence of 
 0.15M KBr and HEPES pH 7.0. Since there is no homologuous structure present, 
 I intend to perform heavy metal derivatization. I read some literature which 
 suggested that I could carry out quick soak with 0.5M Sodium iodide. I was 
 wondering if I could soak my crystals with a similar concentration of NaBr 
 and collect some low resolution data at the in-house source? In addition 
 should I try to also introduce other heavy metals? it might be worth 
 mentioning that my protein carries no methionine/cys residues.
 I have no prior experience in doing this. So, it would  be of great help if I 
 could be directed towards literature/protocols pertaining to this. Any 
 advice/suggestions would be greatly appreciated.

 Thanks in advance
 --
 Amit Sharma

One solution can be soaking or co-crystallization with lanthanide complexes.
More information available in these publications (PubMed):
PMID: 15272192
PMID: 14573945
PMID: 12499547
PMID: 18350532

Re: [ccp4bb] MolRep of coiled coils

[M T]

My short experience with coiled-coil is that molecular replacement can
be difficult for classical software (due to the very anysotropic
shape of the protein).
In our case (a short parallel dimeric coiled-coil), molecular
replacement trials using AMoRe or MOLREP were unsuccessful. We solved
the structure using EPMR (evolutionary search molecular replacement
software).
http://www.msg.ucsf.edu/local/programs/epmr/epmr.html

Michel.

Re: [ccp4bb] Tricks to solubilize protein

[M T]

Hi

The low pH and its interaction with PIP1 can be conflicting. The low
pH can modify the interaction site.
Nevertheless, one good way to study protein stability is thermal shift assay.
You can read this publication too : PubMed ID 16604423. (Rapid
determination of protein solubility and stability conditions for NMR
studies using incomplete factorial design).

Michel.

Re: [ccp4bb] Codon Optimized Expression

[M T]

One classical way to optimize expression level is to screen culture conditions.

For my proteins, I solved my expression problems by changing the
expression vector to a pET or changing a pET 20 to a pET 30 (if the
protein is toxic).

But keep in mind that a low but folded expression is better than a
high expression to inclusion body.

Michel.

Re: [ccp4bb] protein expression problem

[M T]

 I have been trying to express a rat protein in bacteria. The MBP-fusion
 expressed at very high level (~ 40 mg/L) while the GST-fusion and His-tag
 only gave inclusion bodies. The problem is that all protein runs in the void
 volume on a size-exclusion column (s-200, hepes, pH 7.4, 200 mM NaCl), no
 matter it is the intact MBP-fusion or cleaved sample. There is no Cys on
 this protein so there is unlikely any disulfide bond related problem.
 Anything I can do before I throw away this construct and try insect or
 mammalian cells? Thanks.


First of all, using a carrying protein (like GST, MBP) can be disconcerting.
These proteins are very soluble and can solubilize an insoluble protein in
testing condition. So you have something soluble but your protein of
interest can be misfolded or can precipitate when the carrying protein was
cleaved. So keep in mind that a soluble carried protein is not always a good
protein.

After this consideration you have a wide range of conditions to test.
- Solubility of your MBP-fusion: ultracentrifugation, thermal shift assay
(in different pH, salt, salt concentration), micro-dialysis
- Folding of your MBP-fusion: circular dichroism, 1D NMR (if you can,
compare with MBP alone)
- Aggregation/monodispersity of your MBP-fusion: DLS (in different pH, salt,
salt concentration)
- For gel filtration assays don't forget that MBP can dimerize

The second part of your tests can be expression conditions. Sometimes low
but native expression is better than high but carried or insoluble
expression.
- Medium
- Temperature
- Host cells (different E. coli, yeast, insect cells...)
- Inducing strength
- Co-expression with ligands or chaperones

And the last but not the least part of your tests can be refolding.
Inclusion body expression is the first step of your purification. If you
have his-tagged protein in inclusion body a one step purification can be
performed in denaturing conditions. A wide range of refolding conditions can
be tested:
- Flash dilution
- Dialysis
- Refolding by slow gradient on an Ni column (if you have an his tag)
- pH, salt, detergent conditions
- Chaperones
OD at 340 nm can monitor the refolding efficiency.

Good luck

Michel

Re: [ccp4bb] highly soluble proteins

[M T]

First i think that your protein concentration is to low... A common rule is
: protein concentration is good when 50% of your conditions are precipitates
and 50% are clears drops. If most of your drops are clear, i think that you
must increase your protein concentration (a screen from Hampton can help you
for the concentration of your protein: PCT screen).
For your oily bubbles, it often appear when PEG is in the condition, it's
just phase separation.
An other suggestion is that your buffer concentration is too high... If your
buffer is a strong buffer, the conditions buffers can be too weak versus
your 50mM. By the way, real pH in your condition may be something between
the pH of the condition and the pH of the protein sample and not the real pH
of your condition. So you don't have a real access to all the pHs of the
screens.

In summary, reduce your protein sample buffer concentration (to 25 or 10mM)
and increase your protein concentration (to 50, 100 mg/ml or more).

Michel.

Re: [ccp4bb] DNase inhibitors

[M T]

Hi

Is DNAse essential in your protocol? Maybe it's better to not add DNAse if
you are worried about its presence. If you really need to brake DNA, using
sonication may be enough.

Michel

2007/7/31, Dima Klenchin [EMAIL PROTECTED]:

 I have small crystals of protein DNA complex.  I am worried about the
 possibility of presence of some
 DNases in the drop (I use DNase in the lysis buffer). I have Tris and
 MgCl2 in
 the crystallization condition,
 which makes me hesitant to use DEPC or EDTA/EGTA respectively. Are there
 any
 other DNase inhibitors
 that I can use to protect the DNA? Any other precautions one should take
 while
 handling DNA

Re: [ccp4bb] FPLC vs Duo Flow

[M T]

Hi,

In our lab we have background on both instruments, 2 Biorad and 2
Aktas. We got problems on both, minor ones on Aktas, major ones on
Biorads, with expensive fixing on Aktas and cheaper fixings on
Biorads, but efficient fixing on Aktas and unsolved problems on
Biorads. Actually, first Biorad was trashed, second one is a wheezing
old man and Aktas are running like Swiss clocks.
It may be important to note that the Biorads are older than Aktas.

Summary of OUR background: Biorad is cheaper (buying and maintenance)
but have low reliability.

Michel.

2007/7/20, Filip Van Petegem [EMAIL PROTECTED]:

Hi,

I believe this subject has been touched briefly before, but does anyone have
any strong feelings before or against using an Akta FPLC/purifier versus a
Biorad Duo Flow?  The Biorad Duo instruments are significantly cheaper; are
they however also 'as good' as GE Healthcare? I'm especially interested in
comments from people who have used both instruments before.

Cheers

Filip



--
Filip Van Petegem, PhD
Assistant Professor
The University of British Columbia
Dept. of Biochemistry and Molecular Biology
2350 Health Sciences Mall - Rm 2.356
Vancouver, V6T 1Z3

phone: +1 604 827 4267
email: [EMAIL PROTECTED]

Re: [ccp4bb] freezing Ammonium Nitrate

[M T]

2007/7/12, Gebhard Schertler [EMAIL PROTECTED]:




 I have obtained some promising X-tals in 2M Ammonium Nitrate. I am now looking 
for freezing
conditions. Has anybody had experience in successfully freezing X-tals under 
similar conditions
? What was the best cryo-protectant and which concentration was optimal ?


Hi,

You have some informations in supplementary methods of this ref:

Structural insight into antibiotic fosfomycin biosynthesis by a
mononuclear iron enzyme
Luke J. Higgins, Feng Yan, Pinghua Liu, Hung-wen Liu and Catherine L. Drennan
Nature 437, 838-844 (1 October 2005)

Informations are in the first doc at this page:
http://www.nature.com/nature/journal/v437/n7060/suppinfo/nature03924.html
30% xylitol, 2.0M ammonium sulfate, and 100mM  Tris-HCl pH 8.0

Some other good cryoprotectants are small PEGs, glycerol, MPD...

Michel.

Re: [ccp4bb] How to remove nucleic acid contamination for crystallizing zinc finger protein

[M T]

Hi, I think that a good choice, if your protein accept, is to try a gel
filtration at high salt, with or without DNAse incubation before gel
filtration. If your FPLC can do it, a monitoring at 280 and 260 nm will help
you.

Michel