Dear All,
I am solving a ligand-bound protein crystal structure. The ligand is binding covalently to an active site cysteine. However, this cysteine can have 2 orientations in accordance to electron density map which is dependent if the ligand is bound or not (yes, ligand has low occupancy). I would like to create a link only to one of those 2 conformers, however, it is automatically connected to both of them. After refmac the position of the protein-bound part of a ligand is changed so that the distance to sulhpurs of both cycteine conformations is the same (on average).
Is it even possible to do such a thing?
Best
Przemysław Golik
Hi Przemysław,
If I understand your problem right, REFMAC thinks it is the same residue in two different conformations instead of two totally different residues in partial occupation.
Have you tried to build only the relevant conjugated cysteine and see how that behaves in Refmac?
If you simply created a bond between the ligand and one of the cysteines the naming of the two confomers should remain the same with an added alternate conformation 'A' and 'B' and this does require the bond to remain intact for all the species. I think what you need here is two different residue names for the refinement programs to treat them separately.
What you (probably?) want to do is to generate a special residue, your ligand bound cysteine, and introduce that into the peptide chain. There might be some modern and elegant solutions for you if you still want the second unbound cysteine to be included independently in the refinement, I am not certain how to do that yet without trying myself. There is however a more archaic and outright brute way to handle this, which is simply refining two separate protein models at half occupancy with one having the ligand and the other one not. I suggest you don't do that with the coordinates you would like to publish.
The best way to deal with this problem is in my opinion a new dataset with higher ligand occupancy.
Best,
Stefan
Hello and thanks a lot Stefan Gajewski , I will give it a try.
Best,
Przemek
Another simple but crude method you can do is open the coordinates in coot with density map. Click on any one of the following tools in the right hand panel of coot: 1) Real space refine or 2)Rigid body refine or 3) Rotate translate zone and select your residue and the ligand to be connected. Most probably one of the first two should work. Sorry I don't remember exactly which one but had worked for me.
Hi,
The question is too old, but you did not get the answer.
The proper way is to generate two conformers for your cysteine (in Coot). Then one alternative conformer (A) connect to your ligand. Ligand should have partial occupancy matching the occupancy of the cysteine to which it is connected to. The ligand has to have the same alternative conformation, in your case A, since it will be connected to A conformer. This way REFMAC will not link your ligand (conformation A) with your cysteine in conformation B.
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