Hi everyone,
I’m refining an I1 symmetry particle and trying to resolve assymetric subunits after symmetry expansion and subparticle extraction.
My workflow is refine with I1 symmetry, symmetry expand(I1 sym), shift coordinate centers with volume align tools, reextract with smaller box size(subparticles of assymetric unit), then cryosparc’s 3D classification job(~12 million particles). The 3D class job does well with my downsampled particles and looks pretty decent but when i take the selected classes that i want and move them onto refinement(~1 million particles) jobs they turn into blobs that resemble noise. Are my subparticles just too noisy?
Please let me know if any other further info is needed and any advice would be greatly appreciated.
Best,
Tyler
Is this local refinement? Can’t do global refinement on symmetry expanded data.
Try severely restricting the angles and shift ranges; by default even Local Refinement gives too much range and the particles contributing to views “through” the capsid will drift off position and make everything a mess (block based reconstruction is something I still do exclusively in RELION partially for this reason…)
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Hi,
@tbrittai how do the heterogenous reconstructions for these classes look? Additionally, as @rbs_sci mentioned, only use local refinement and limit the search extent to something like 10 degrees and 3A shift or 5 degrees and 1A shift might be beneficial. Using the gaussian priors for pose/shift search might also be beneficial.
@rbs-sci, do you have issues attempting to do block-based reconstruction in CS? Additionally, how often are you performing this type of processing workflow? Only for large complexes/virus capsids?
Best,
Kye
My lab does a lot of work on giant viruses, so fairly frequently. Yes, normally only for larger things, although we did test it for a smaller virus with a highly flexible binding domain. Whenever I try working with giant viruses in CryoSPARC, particularly BBR, the blocks always end up very streaky “through” the capsid, regardless of the direction the capsid block is facing in relative to x/y/z. Sorry, I don’t have any current examples to share. 
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Curious - do you see less streaking/anisotropy with Relion for these large capsids?
Yes.
It’s “easy” to track as refinement proceeds; the angular distribution plot drifts off from a nice symmetry expansion pattern - even in local refinement - to a less uniform distribution where views are dominant polar/equatorial.
Severely restricting the angular search and shift ranges help a lot, but it’s still not as clear. Having said that, I do wonder if it’s partly the convergence criteria; CryoSPARC converges a lot faster and ceases iterating angular sampling by default at 0.2 degrees in local refinement (although I’ve changed this to 0.1 and 0.05 degrees without seeing much improvement), which is an order of magnitude less fine than RELION usually converges at for whole capsid and BBR of giant viruses… I pretty consistently have the refinements in RELION converge at 0.02 degrees across multiple datasets.
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