I am trying to follow the tutorial case Yeast U4/U6.U5 tri-snRNP to learn local refinement. I have two questions regarding the tutorial:
1, when you use the mask to subtract the region you don’t want to refine, do you use the dialated mask to do the subtraction or do you use the original generated in chimera to do the subtraction.
2, about the search range: in the tutorial, it says ‘based on the range of motion seen in the particle from the heterogenous refinement earlier’, I am wondering where in the heterogeneous refinement can I find these parameters.
3, I did the local refinement on the head part, and tried to follow the tutorial, and I got a 4.13Angstrom structure for the head part which is lower than the one the tutorial got. I am not sure what could be the reason or what I can do better.
You should likely use the dilated mask, since any masking operation that uses a sharp mask will create artefactual high-resolution edges in the masked volumes that then get projected for subtraction. It is okay to have some overlap between the mask used for subtraction and the one use later for local refinement.
The search range unfortunately can not be read out from any other job - it depends on the true range of possible motion of the selected region of the molecule, and only in some cases will heterogeneous refinement be able to isolate a map at both ends of the motion range. So in many cases, you would have to guess or try multiple local refinements with this option.
Unfortunately I can’t diagnose the discrepancy without seeing the jobs, but local refinement is quite sensitive to parameters and the choice of masks/dilation as well, so perhaps trying with a few other choices may yield a similar result?