r/Biochemistry • u/SufficientAd3103 • 11d ago
Small-molecule crystallographer working on a protein structure
The frustrating part is that I understand the process in theory, but I am not familiar with the software so it takes me forever. I am currently having difficulties with 2 things:
- Molecular Replacement gives me a solution where the peptide is broken, so Coot reads it as 2 chains rather than one. Why does this happen since a sequence was given? Can it be fixed?
- I have to modify a residue and I can import it using the 3 letter code but Coot places it in a conformation that doesn't make much sense, I want to flip it so that it goes on top of the electron density blob but I keep failing at this.
Are you aware of any resources, such as a mailing list or a forum, where one can ask questions and people can exchange information, about Coot or other protein crystallography programs? I have already read the Coot manual and checked their youtube channel but found no solution to my 2 problems.
EDIT: You are incredibly helpful, thank you so much. I will respond asap ( weekends are usually hectic for me). I am using Coot 0.9, ccp4i2, Molrep, and Refmac5. I would like to use Phenix but I don’t have a Mac and it keeps crashing on PC.
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u/PossiblyUrChemTA 11d ago
If it's supposed to be one chain, change the chain ID so they're all the same.
When you're choosing your residue range to drag in the modified residue, make sure you're selecting residues +1/-1 to the one you're trying to move. You need to give the backbone room to move.
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u/SufficientAd3103 8d ago
I have been using "edit" --> "replace residue" and I center on the exact residue I want to change. I have never seen the "residue range" option, where can I find it?
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u/PossiblyUrChemTA 8d ago
When you click real space refine, you have to define the zone you want to refine with two clicks. Make the zone 3 residues, centered on the one you want to fix, instead of just selecting the one you want to fix.
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u/Miciussd 10d ago
There is CCP4BB mailing list. Refining protein structure takes time.
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Which program for MR did you use? Most (if not all) MR programs just place your model desired number of times. If something is missing then either your model was missing or you did MR with model building and it just truncated it.
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If you want to modify the residue you should use the panel on the right side of coot window (the "atomic" sign, yellow and black) to mutate the residue. If you add a molecule via its 3 letter code it is added to different molecule, you need to tranfer it to the main molecule through edit options (top bar, edit merge molecules).
If you want to insert some unnatural amino acid and make it a part of peptide chain, then you need to make sure that its definition is correct, and that it is not a monomer in its description.
Płacing something in a blob can be done through refine option - top right corner blue ball. Then you can nudge the molecule into its place with you mouse.
Those are rather basic utilities of coot - you should be able to do that once you finish coot tutorial (either 0.9 or 1+ version e.g. https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/web/tutorial/coot1-tutorial1.html)
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u/SufficientAd3103 8d ago
Would you recommend fixing outliers and other issues before and after modifying or modifying a residue first and then fix all the other issues?
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u/Miciussd 8d ago
Refinement is an interative process. You can do many things at the same time. And then do them again. And again. And...
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u/xtalgeek 10d ago
Let's back up. What did you use as an MR model, what program did you use to perform MR, and did you attempt auto building after MR? Making poor choices in these steps could result in disconnected models.
It usually pays to inspect the MR model conformance with electron density after an initial refinement, and verify that crystal packing makes sense before doing sequence corrections in the initial model manually or performing auto building.
After getting an acceptable MR solution, rebuilding and refinement takes quite a bit of time and effort: peptide chain corrections first, cofactors with high electron density (metal ions, etc.), then solvent, cations, and anions if present. It's a slow process to improve Rfree and model integrity to an acceptable point. Automation is not always the quickest way to get there, especially for low resolution data, or for targets with stretches of relatively disordered regions, or for marginal but acceptable MR models. In the latter cases, poorly defined regions clean up significantly once the rest of the model is conforming and phases become more accurate. Auto building too early can just make a mess.
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u/SufficientAd3103 8d ago
MolRep within ccp4i2, I did not attempt auto building that I know of. I used a .pdb file downloaded from the PDB that I was told is the closest to what they expect their protein crystal to be and has the same residue change. Unit cell and SG match.
I understand it takes time! I'll be patient!
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u/xtalgeek 8d ago
Phaser is much more robust than Molrep. Also check your MR model to see if it has chain breaks. MR should not break chains during phase solution. You don't have to search with a model from the same space group. You just want a quality starting model. All non protein elements should be removed from your model. And in some instances, one might trim back disordered chain termini as well.
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10d ago
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u/SufficientAd3103 8d ago
R = 0.29 Rfree= 0.33
Haven't tried the alphafold yet.
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8d ago
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u/SufficientAd3103 8d ago
I was able to renumber them and to close the chain by doing a "refine chain". The model doesn't have the gap.
No worries, I truly appreciate the help!
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u/East_of_Adventuring 11d ago
Not sure how much help this will be but a couple points:
Do you refine after doing molecular replacement or take it straight into Coot? I would refine first, mess with the weights of the density versus the chemistry (there are good guides online for this). Also check your molecular replacement structure is intact and that nothing is weird there. Do you have a good Alphafold model? That can be used for molecular replacement instead of an actual solved structure. If you're already doing this, try the reverse and work with a similar published structure.
You can manually move residues around with one of the default Coot tools on the sidebar. If you're finding difficulty refining this, try and just move it into position and let Phenix take it over from there. Also, CCP4 has alternative refinement tools and some ligands or structures are going to work better for certain applications. You'll need to try a few different things. One more thing, Coot can regularize and factor only the chemistry into a refinement. This is useful when you are confident your residue is there but the density might be a convincing the weighted refinement to do weird things.
You'll get more comfortable with time, so try not to stress. I know it can be controversial, but I actually find Google AI great for teaching me how to use these softwares and answering questions like "where do I find the button/setting for this function?"
Good luck!