preserving weak density during refinement

[Madeline Rollins]

Hi all,

I recently ran a classification job (e2spt_refinemulti_new) for my dataset. One of the classes has the membrane-bound ribosome and another weaker membrane-bound density. I would like to further refine this class without losing this weaker density – because the ribosome and the membrane have a much higher signal-to-noise ratio, I think they dominate the alignment, so the weaker density disappears over the course of the run. Does anyone have any advice on how to preserve this weak density in the downstream refinement runs? 

 

My e2spt_refine_new job is set up as follows:

 

e2spt_refine_new --ptcls=sptcls_01/aliptcls3d_10_00.lst --ref=sptcls_00/threed_10_00.lst --startres=25.0 --loadali3d --goldstandard –sym=c1 --iters=p,t,t,p,t –keep=0.9,0.5,0.5 --mask=masks/cylinder.mrc --localrefine --parallel=mpi:64:/scratch/beagle3/mgrollins --threads=64 --m3dthread

 

Would it help to include --maxang to define a maximum angle difference for the localrefine parameter? Or would it be better to change my masking strategy? I tried using --maskalign with a spherical mask around the weaker density and some neighboring components that are a bit stronger, but it didn’t seem to help.

Thanks,

 

Madeline

[Steve Ludtke]

If one part of structure gradually fades as the contrast of another part improves, that clearly implies motion/variability. If you take the particle subset from the iteration where the density still exists, and do a further classification on that, or use e2gmm.py or other variability analysis tools, you may be able to extract a more homogeneous subset, but that would require that you have sufficient particles to start with. You may also be able to do a focused alignment or classification to improve your results. While it's mathematically a little iffy, using a mask where the mask value in the higher density regions you wish to de-emphasize is lower than 1.0 can also help. For membrane patches in-situ which can have a lot of local variability in ways which influence overall alignment, you may also consider restricting the mask in that region to a small cylinder around the area where the ribosome is bound.

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