Symmetry relaxation with 3D classification

3D Classification
1

Hi everyone,

I have a few questions regarding a symmetry-breaking workflow in CryoSPARC. I am analysing a C24 symmetric assembly and would like to determine whether any individual monomer contains additional density.

My initial idea was the following:

  1. Refine the structure with C24 symmetry.

  2. Symmetry-expand the particles to obtain N× more particles.

  3. Apply a mask around a single monomer and run a classification focused on that region to check for extra density.

However, I noticed that the 3D Classification job in CryoSPARC does not include explicit symmetry options, so I am unsure whether this is the correct approach.

Do you recommend a more appropriate workflow for symmetry breaking?
I considered running a NU-refine job with symmetry relaxation enabled, but I am not sure what the recommended next steps are after symmetry relaxation. My understanding is that one should typically perform a global refinement after symmetry expansion, but I’m not completely certain how to combine these steps for focused classification.

Any advice on the best way to proceed would be greatly appreciated.

Thanks in advance,
Chencho

2

The workflow you describe is fine, I would try the Class3D approach before attempting sym relaxation.

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3

I agree with @olibclarke’s comments. Never had that much luck with symmetry relaxation, although that might be sample related.

Symmetry options are definitely there in CS 4.7.1:

Screenshot from 2025-12-10 08-35-11
Screenshot from 2025-12-10 08-35-112095×415 83.2 KB

This is absolutely something you should not do after symmetry expansion, as it risks the expanded particle set multiplicates aligning against the same view, artificially inflating resolution or worse. After symmetry expansion, local refinements only. Global CTF refinements are OK, but that’s not volume refinement, but electro-optical parameterisation, and a different matter. :slight_smile:

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4

They are there - but (I know you know this but just to clarify for the OP) you shouldn’t use them for this purpose, when you are trying to identify deviations from symmetry. Sym expansion followed by classification in C1 is the way to go (or at least the first thing I would try)

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5

Yes - agreed! When searching for potential asymmetry in a (nominally) symmetric complex, symmetry expansion and C1 3D classification is always my first choice also.

I could have been clearer, although when I read “3D classification does not include explicit symmetry options”, I did briefly wonder whether I’d been dreaming it! :rofl:

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Thanks for the comments. When I said that I do not see symmetry options in the 3D classification job, I meant symmetry-relaxation options.

Best

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7

Yeah sure, you are totally right. Thanks for the tips.

I will try definitelly Sym expansion + 3D classification

Best

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