FSC not reaching zero after symmetry expansion

Data Processing Strategy
1

Hello!

I am currently processing a dimeric complex and am encountering a problem after symmetry expansion and re-extraction of the particles.

I first refined my complex in C1, using homogenous refinement, followed by an homogenous refinement in C2.
Each monomer have strong conformational heterogeneity, so I proceeded with C2 Symmetry Expansion, and another round of homogenous refinement in C1 with a mask covering only one monomer.
I then shifted my particles using the Volume Align Tools to recenter the particles on only one monomer, and performed another round of homogenous refinement in C1.
Then I reextracted the particles (duplicated), refined them again, and now i am getting weird FSC curves that won’t cross the 0 line …
Like this:
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I guess this comes from the duplicated particles and probably also half-set assignment …

Did i do something wrong?
How would you proceed with that?

Best,
Flo

2

This is expected. When you perform symmetry expansion, you are effectively duplicating your particles but changing the orientation of the copies.

This won’t cause a problem in local refinement (as the orientations of the copies are different, you won’t get spurious correlations), but it will cause a problem in homogeneous or NU refine, where complete orientational search is performed.

I would suggest performing local refinement immediately after symmetry expansion (and you can then perform subparticle extraction if you wish).

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3

As Oli says, if you symmetry expand, you have to stick with local refinement. Global refinements will do exactly what you’re seeing.

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4

Thank you both ! Indeed only using Local Refinement fixed the problem.
Captureatp3

Best,

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5

Hi all,

Just chiming in; as of CryoSPARC v4.5 released May 7, 2024, refinements with global pose search will raise a warning if symmetry expanded particles are detected in the input particles.

Best,
Michael

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