Hi Careina,
I can think of two possibilities:
- your restraints are not strong enough.
- if your mutant dataset is of significantly lower resolution than the native, almost any refinement will make the model worse. If this is your case, just change the mutated residue and very, very obvious things in the difference map.
Then refine with strict restraints, perhaps only refining overall B and only refining the position of the mutated residue and any other obviously changed residues.
If possible refine restraining to the native structure (I only did this a long time ago with TNT with a script Jan Pieter Abrahams wrote, I don't know if refmac has this possibility).
greetings,
Mark
Mark J van Raaij
Laboratorio M-4
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
c/Darwin 3, Campus Cantoblanco
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://www.cnb.csic.es/content/research/macromolecular/mvraaij
On 22 Jun 2011, at 08:25, Careina Edgooms wrote:
> Dear ccp4 members
>
> I have something that surprises me with molecular replacement. I have obtained a solution for a single point mutation using Phaser, the solution seems ok. I do one round of refinement with refmac and I check the structure using molprobity before I even start really to refine it. The structure looks very good. MolProbity gives all green lights. Then I start to "fix" the structure, I add waters, I check fit to density, rotamers, geometry etc, I do some more refinements. I check with MolProbity and it looks a lot worse... many clashes, many bad ramachandran and rotamers, many red lights. I do not understand. How can each successive round of refinement make the structure worse and worst? Is there a fundamental problem, perhaps, like undiagnosed NCS or incorrect space group or incorrect MR solution? Could they be giving these strange result do you think?
>
> Thanks
> Careina
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