Also - during local refinement I often see a population of particles that retain large shifts in both orientation and offset through the final iteration (see below) - they don’t seem to converge, whereas the rest of the particles do. It might be nice to have a way to select and test the removal of such particles, as I suspect they are garbage.
Cheers
Oli
@olibclarke yes, that is how the dynamic masking should work.
We’ve also noticed the population of particles that seem to drift far from their original alignments in a few datasets. We will work on something to filter them out!
Best,
Ali
Doesn’t seem to be quite working as expected though - see PM
@ali.h most of what you said makes sense (I like the dynamic-static mask approach as well), but I am not so sure about the fulcrum position.
I think you should draw pictures of a stack of several particles with a rigid body displacement, centered on the CoM or the fulcrum, and think about how the whole cost function will evolve over a few iterations in either case. It’s ultimately an empirical question, but there is a reason why CoM-centered refinements have been preferred throughout the history of single-particle analysis. Perhaps branch-and-bound changes the calculus since fine global search can be made reasonably fast.
With local refinement I indeed get an improvement of my volume. Could someone comment on these plots, and what parameters (from default) would you adjust ?
I have a small membrane-protein complex, box size is 338.
Number of particles is around 55000 for this job.
Thanks a lot for you help !!
Jacopo
I am now attempting inputting coordinates for the placement of the fulcrum. Is there any other best practice over what is described here:
https://cryosparc.com/blog/local-refinement-snrnp-case-study/
@olibclarke
Hi Oli, Amazing improvement by local refinement. Congratulations! After examing all the messages, I’m more confused about where to set the fulcrum. I’m wondering in this case, did you set the fulcrum at the center of the mask or the junction between the bodies? Thanks, -Rui
Hi Rui, I set the fulcrum at the center of the mask.
Cheers
Oli
Hi Ali,
Is the symmetry option avaiable for local refinement now?
There is no option to enforce symmetry in local refinement. However in the meantime, performing symmetry expansion prior to local refinement will at least give you the advantages of averaging the individual asymmetric units.
Cheers
Oli
Hi,
This thread was a life saviour. When one does particle subtraction, should the input volume for local refinement be the volume from NU-refinement with full particles?
Best regards,
Nuno
Hi @nbustorff,
Yes - you can input the volume from a NU-refinement that used un-subtracted particles to a local refinement job. When doing this, the mask supplied to the local refinement should cover only the region of the volume that you are interested in refining (thus, it should only cover the portion of the volume that wasn’t covered by the mask used in the particle subtraction job). Let me know if this made sense!
Best,
Michael
A quick workaround in the meantime is to open the cs file, set alignments2D/class_posterior to 0 for particles that don’t converge, save and re-import the particle gorup. You can then use probability filter job to remove drifting particles from the imported particle group.
