Hello, everyone! I am a rookie to Cryo-EM data processing. I encountered a problem that I got a good class of particles after Ab-Initio Reconstruction but the density became worse after NU refinement. The parameters for NU refinement were default except that the Initial lowpass resolution (A) was changed to 10 Å. Any ideas about that?
the map colored by yellow is the initial model and the gray one is the map after NU refinement.
This looks like a very small molecule. In such cases, the dynamic masking procedure in NU-refine often makes things worse - I would suggest switching it off (by setting the start resolution for dynamic masking to 1Å).
Alternatively, you can try initiating local refinement starting from the poses you have generated with ab initio.
Hi @olibclarke ,
Thank you for your suggestions. I have tried to set the start resolution for dynamic masking to 1Å during NU-refinement but got similar result as before. I also tried local refinement using the initial model and corresponding particles without any improvement on map density. I wonder whether the strong micelle signal will hamper the resolution and how to solve this problem?
Best,
Verdandy
Hmm - how many particles do you have remaining? It may also help to try to clean up your particle set more, e.g. by heterogeneous refinement with one “good” model and several junk classes
Hmm - the density shown looks much smaller than 140kDa - maybe some domains disordered? If the protein is a symmetric dimer, have you tried enforcing C2 symmetry in either ab initio or refinement?
No, it looks small in terms of the number of structural elements present. Looks like the equivalent of ~2x7TM bundles, with nothing outside the membrane.
Hi Oli,
I have met the same question with Verdandy AND I just tried your method which is initiating local refinement starting from the poses you have generated with ab initio.. However, it always failed and showed I should put some half sizes. I try two kinds of local refinement, but both failed. So, is anything wrong with my operation ? Thank you.
Qu
Hi Oli
I guess I get your point. The local refinement must be done after Homo/heter refinement. So the mask should be put from the homo refinement. Thanks a lot.
Qu