[ccp4bb] Real space refinement of alternative conformations
This is a cross-post from the cootbb last week, regarding the modelling of multiple conformations in coot. I know this has long been a tricky thing to do, and in the past I've always managed to model multiple alternative conformers by hook or by crook, but in some current structures I'm having a bit of a nightmare getting things to refine with reasonable geometry, and was wondering if anyone has noticed this and/or managed to resolve it satisfactorily. If you add an alternate conformer in coot, you cannot then do real space refinement with either sphere refine or real space refine zone (eg i +/- 1 by pressing 'a' or by selecting a zone around the residue with multiple conformers). In the case of sphere refine, the i-1 backbone peptide will become non-planar (or completely flip so the C=O bond overlaps the C-N bond), and the i peptide will also be distorted though to a lesser extent. In the case of real space refine zone, the i-1 and i backbone angles are distorted such that C-N-CA is around 150 degrees. You can real space refine zone, then just select one of the multiple conformers, which has worked acceptably in the past (especially when then fed into a refinement package, which tends to clear things up), but at least on the several datasets I've tested it on, this is still leading to distorted geometry, particularly peptide planarity of the i-1 and i peptides. I'd really like to get a model with good geometry directly from coot, so I don't have to rely on maximum likelihood refinement to sort out the problems - I always find that a rather clunky solution. Has anyone else noted or documented this behaviour, and is it possible to fix this? I know I may be asking a lot, here! To clarify some of the obvious questions up front: I'm using coot 0.8.1 on Ubuntu 12.04 and coot 0.8 on Max OSX with the same results. The observed behaviour occurs with and without the following restraints in coot: planar peptides, torsion and Ramachandran restraints, with a refinement weight typically of about 10, but I've observed the same problem at any value between 0.1 and 90 (I haven't tested wider as there's not much point!). You don't need high res data with genuine alt conf density to test this, you get the same behaviour by modelling in 2 x 0.5 occ conformers into the same density. The only work around so far seems to be real-space refining without the second conformer to get all the geometries right, then putting in the alt conf and selecting the nearest rotamer, then do ML refinement to try to fix the errors and get B-factors. Deleting the alt conf in coot before any additional real space refinement is then required (but coot cleverly remembers the B factors for the alt conf when you add it back in, which is neat). I'd be extremely grateful for any help to resolve this! Thanks in advance Isaac Westwood
Re: [ccp4bb] Superposition of select residues
I find that the superposition in CCP4MG is the most flexible and easy to use implementation of any program I've used. There are multiple superposition methods each with the ability to select residues or atoms. It's very powerful. CCP4MG also happens to produce very pretty pictures! Isaac On 5 Feb 2015 22:41, Kgosisejo, Oarabile o.kgosis...@usask.ca wrote: Hi all, Does anyone know of a program to use to superpose only selected protein residues, e.g. enzyme active site residues. I solved my structure using molecular replacement now I want to compare its active site to those of homologous structures. I have used the DALI server before but I had to upload the whole model. Thank you for your advise *Best Regards,* *Oarabile M. Kgosisejo, MSc. Candidate University of Saskatchewan * *o.kgosis...@usask.ca o.kgosis...@usask.ca*
Re: [ccp4bb] Protein-Ligand Crystallization
In addition to the previous suggestions, if you have a metal-binding protein, beware of acidic compounds chelating the metal and stripping it out of the protein, as this often lead to the effect you observed as well. Isaac On 5 Feb 2015 13:45, Monica Mittal monica.mitta...@gmail.com wrote: Hi all, I am working to crystallize a protein-ligand complex. I did a preliminary melting curve analysis for the protein in the absence and presence of 2 ligands (dissolved in protein buffer). I kept the other controls as buffer an a known standard to confirm instrument performance. All expts done in triplicates. Now the results are like : Tm of protein alone is 56 deg, Tm in the presence of Lig1 conc. of 0.05mM, 0.1mM, 1.0mM and 10.0mM is 56.2, 56.1, 56.0 and 53.5 respectively !! Tm in the presence of Lig 2 conc. of 0.05mM, 0.1mM, 1.0mM, 4.0mM and 10.0mM is 56.2, 56.1, 54.9, 52.3 and 50.6 respectively !! Although the effective delta Tm for both is different at higher concentration, but both are kind of making protein less stable. So i was wondering, will it be difficult to co-crystallize them !! Any suggestions in this regard are highly appreciated !! Thanks Monica
Re: [ccp4bb] water at the same exactly position
Dear Lu Zuokun, There are several methods you can use to locate these problem residues. Here are 3: a) manually step through your PDB file in coot (good practice anyway, this is fundamentally what we as crystallographers should be doing when we are model building) b) identify waters with close contacts as has been detailed earlier in this discussion c) run a round of refinement - if you use buster, it will fail to start and the output files contain full details of all of the close atoms/residues, then you can go through and edit/delete as required in coot (or in a text editor). if you use phenix or refmac to refine, and if the refinement doesn't fail (again, details in the logs will tell you why), then the atoms should refine away from each other during positional refinement. Then on your next round of model building when you step through the entire structure in coot, you'll see this. I've done this a few times due to careless/over-exuberant use of keyboard shortcuts in coot. It's not too onerous to identify and fix these problems, though! Regards Isaac On 30 October 2014 12:14, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Lu Zuokun, it may appear tedious, but for a good quality model you should check every position and decide yourself what to place there. Automation will not replace your chemical understanding. Regards, Tim On 10/30/2014 10:56 AM, luzuok wrote: Dear Nat, But if there are other solvent molecules, such as Mg2+, SO4-,, in the same position. How can this problem be solved? Lu Zuokun -- 卢作焜 南开大学新生物站A202 在 2014-10-30 12:57:31，Nat Echols nathaniel.ech...@gmail.com 写道： On Wed, Oct 29, 2014 at 8:53 PM, luzuok luzuo...@126.com wrote: I think it is better for COOT to solve this issue. Coot already can be used to solve this issue - I think the automation is somewhat lacking, but it's vastly preferable to anything involving a text editor or shell commands. 1. Load molecule and electron density maps in Coot 2. From the Validate menu, select Check/Delete waters... 3. Just select for waters with very close distances, for example 0.2Å; I've attached a screenshot of what it should look like. 4. This will give you a list of overlapping waters - then you just need to delete one of each pair. (It doesn't matter which one - the waters will be renumbered later anyway.) Alternately, you can set Action to Delete, which is much less effort, but that will delete both copies. If you are just going to run a program (or Coot function) to place more waters automatically (my preference), this won't matter, but if they're atoms you really care about, you should delete them manually. -Nat - -- - -- 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 Icedove - http://www.enigmail.net/ iD8DBQFUUiuJUxlJ7aRr7hoRAp+GAJ0f7y+JtPptqADxkFQIoQjOXMuI6QCgj0UB CtBP+UbvdJ7b5pFfUOtp3KA= =yOAl -END PGP SIGNATURE-
Re: [ccp4bb] Unidentified density in coot
Dear Shanti Pal, did you use ethylene glycol as cryoprotectant? It may even be there in small amounts in your PEG400 solution. As Nicholas and Tony have said, this could be noise (or could be distorted due to noise) as it's on a 2-fold axis. From those pictures, it looks to me like one molecule of ethylene glycol (Coot - Get Monomer - EDO). Don't forget to drop the occupancy to 0.5, though! Best Isaac On 23 June 2014 13:17, Shanti Pal Gangwar gangwar...@gmail.com wrote: Dear All I have solved a structure of my protein at 3.0 A. The crystallization condition is consisting of PEG400, NaCl, MgCl2 and Sodium citrate. The protein was purified in HEPES buffer. I can see an unidentified electron density blob in coot and I am not able to figure out what it could be? I have attached the snapshot of that blob with this mail. I request everyone to please help me in identification of this blob. Thanking you in advance. Shanti Pal Best regards Shanti Pal Gangwar, Ph.D School of Life Sciences Jawaharlal Nehru University New Delhi-110067 India Email:gangwar...@gmail.com
Re: [ccp4bb] Invisible atoms in ligands
Hi Tim, The problem I see with including atoms with no density is that the one place you can guarantee those atoms are not (at 100% occupancy), is at the coordinates assigned to them. I would argue that the scientific inaccuracy of such a model should outweigh the desire to use the coordinates for downstream calculations. A better solution may be for the PDB to require us to submit the PDB and CIF file we generate for the full ligand in addition to the protein PDB file which may only contain a truncated portion of the ligand as part of the model. I prefer omitting the atoms in the same way I do for the protein model and for the same reasons. I accept this makes it potentially more troublesome for downstream users as things stand, although would argue that there is greater potential for harm if the unsuspecting user believes the atomic positions are correct, where a ligand (or protein) atom has been modeled into a region with no density. Best, Isaac Westwood On 13 June 2014 11:35, Robbie Joosten robbie_joos...@hotmail.com wrote: Hi Tim, The problem with missing atoms in ligands is that you cannot use the coordinates for any follow-up calculation that requires ligand topology (e.g. restraint generation). That forces you to rely on the annotation of the compound, for instance at the PDB. That can be quite messy and leaves extra room for errors and misunderstandings. Cheers, Robbie -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tim Gruene Sent: Friday, June 13, 2014 12:04 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Invisible atoms in ligands -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Hi Frank, if you can calculate their position, i.e. it is part of a rigid group, I would leave it. If it is flexible, I would remove it because the reader of my deposited structure may not be a crystallographer and misinterpret the result. If it is obvious that some atoms are missing, it is even better because it is more likely to make the reader think about the reasons why part of the ligand is not displayed in the model. Cheers, Tim On 06/13/2014 11:45 AM, Frank von Delft wrote: Hi all - talking about ligands, a quick question on that old conundrum, of what to do about invisible atoms -- build them with occ=0, or omit them? For bits of protein, I know all the arguments; personally I prefer omitting atoms because: * for amino acid sidechains, their presence is implied in the residue name. * for whole residues, their presence is implied in the sequence numbering However: what about ligands? Nowhere else in the PDB file is their presence implied - or have I missed something? (Certainly disorder in a ligand is important information that needs to be captured!) Cheers phx - -- - -- 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 Icedove - http://www.enigmail.net/ iD8DBQFTmsx7UxlJ7aRr7hoRAhxOAJ9YXwdr4kZULidLAdzTgEjdZwfXNQCeKYT A 9vSeeDh7eq2v4CwBII64wn8= =uviV -END PGP SIGNATURE-
Re: [ccp4bb] Calculating volume of Ligands
Dear Rajan, In addition to the estimates already mentioned, if you have access to Chem3D (Cambridgesoft), you can calculate the Connolly volume (excluding solvent) for any (preferably small) molecule, either drawn by hand or by input from its coordinates. In ChemBio3D ultra, this is found in calculations computer properties ChemPropStd. Another guestimate method is to determine the distances between various pairs of atoms in, e.g. Coot, and to do a back-of-an-envelope calculation for the volume based on those, but that's probably less accurate than the estimate methods already mentioned! Isaac -- Dr Isaac Westwood Department of Pharmacology University of Oxford Mansfield Road Oxford OX1 3QT tel: 01865 271595 email: [EMAIL PROTECTED] _ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Rajan Pillai Sent: 15 January 2008 21:40 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Calculating volume of Ligands Hi All, Can anyone tell me any program that calculates voume of a ligand? Moreover, is there also any program that can calculate the volume of a ligand from its coordinates? Thanks, Rajan.
Re: [ccp4bb] Measuring the volume and surface area of an active site
Also VOIDOO is an excellent program for this. HYPERLINK http://xray.bmc.uu.se/~gerard/manuals/voidoo.htmlhttp://xray.bmc.uu.se/~ge rard/manuals/voidoo.html Isaac _ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Rajan Pillai Sent: 13 December 2007 22:45 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Measuring the volume and surface area of an active site Hi All, Can anyone tell me if there is any program available that can measure the surface area and volume of the active site of a protein? Thanks, Rajan. No virus found in this incoming message. Checked by AVG Free Edition. Version: 7.5.503 / Virus Database: 269.17.1/1183 - Release Date: 13/12/2007 09:15 No virus found in this outgoing message. Checked by AVG Free Edition. Version: 7.5.503 / Virus Database: 269.17.1/1183 - Release Date: 13/12/2007 09:15
Re: [ccp4bb] How to determine ligand binding from diffraction pattern?
I'm a fairly inexperienced crystallographer, but I have worked on a few cases very recently (from separate protein systems) in which the ligand was not clear in the electron density until quite a long way through refinement: in the latest case, it wasn't possible to be too confident of the ligand's presence (rather than, say, water) until the R/Rfree were approx 21/26% respectively, and only after several more rounds of refinement were we confident of the orientation (R/Rfree 18/23%). As I said at the top, I'm fairly inexperienced, so I'm not yet sure whether this is an extreme case, but I think it's definitely worth doing molecular replacement and refining as much as possible before giving up on a dataset! The change in electron density quality - especially in the region of the ligand - steadily improved throughout refinement. One thing also worth remembering is that the occupancy of the ligand is not necessarily 1. I was nearly put off a dataset completely because of this, until a considerably more experienced colleague pointed out that it's probably not in every single binding site - after playing around with the occupancy (from 1.0 to 0.85), the model suddenly became very reasonable, which was nice :) Of course, the caveat is not to over-refine... Isaac -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Palm Sent: 29 May 2007 21:25 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] How to determine ligand binding from diffraction pattern? I would like to expand on the question and answer below and compare your experiences: Looking for ligands in many different soaks / cocrystals of your protein of interest, you still should do molecular replacement and a bit of refinement. I agree with Steve, but how much refinement is necessary and enough? We have a specific case with a 24 kDa protein crystallizing in P6522 with resolution of 2.5 - 3 A, which should be comparable to most cases. The ligands have 10 - 20 non-hydrogen atoms (most of the time we don't know, we are actually screening for them). How far should we refine to see if we have only water molecules or a ligand bound - to an Rfree of 0.45 or 0.40 or 0.35? greetings Gottfried Dear all, Is there a simple way to determine whether ligand is bound or not by comparing the diffraction patterns between ligand-free (structure known) and ligand-soaked protein crystals? I would like to solve the ligand bound protein structure, but before I do so, I have to find out if the ligand is actually bound. Thank you very much! Best, Joe Having done this a few hundred times, I would strongly suggest that you just collect the data and solve the structure. Since you already have the apo structure solved, then it really isn't that much work to do an MR solution on the complex. Be aware that quite frequently there is enough non-isomorphism to necessitate partial refinement of the complex structure before recognizable density will appear for the ligand. The definitive answer can only be obtained with a full data set, so go for it. Good luck- Steve No virus found in this outgoing message. Checked by AVG Free Edition. Version: 7.5.472 / Virus Database: 269.8.1/822 - Release Date: 28/05/2007 11:40
