Symmetry question

3D Variability Analysis
1

hi all
I’m unclear on how to approach 3DVA when I have sym. My protein is D2 sym: it has four monomers, each of which is 60kda. I refine in D2, and it is large enough (240kda) to run successfully. Now for 3DVA do I…

  1. Use a mask of all 4 monomers and do the 3DVA in C1?

  2. Expand the D2 sym and do 3DVA with a mask around a monomer. I think this is ideal, but 60kda is not a lot of signal for 3DVA I dont think?

  3. re-run the refinement in C1 and for 3DVA use a mask around the full tetramer?

thanks!

2

2 I would say - I think 60kDa should be enough signal

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3

thanks for response. I ended up trying all of them (so fast in cryosparc). Was just hoping to have the best approach beforehand so I can have the most confidence in the results…

approach 3 gave the biggest (and somewhat expected) conformational change. approach 2 was a bit less informative but may be useful for diversity of the ligands. Still trying to figure out what it all means.

thanks!

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4

@orangeboomerang hi, i was wondering why you re-ran refinement in c1 before running 3dva? wouldn’t you want your particles aligned to D2 symmetry convention before running 3dva…?