I have a stable homodimeric protein (mass photometry, size exclusion etc.), which has been crosslinked, which produces a structure of around ~8A.
The 3D classes are the volume of a dimer, and show aspects of symmetry ab initio - but I could be convinced otherwise. If I apply C2 symmetry in the homogenous refinement , I produce a higher resolution structure, which makes more sense biochemically etc.
My question is, how do I justify “forcing” the C2 symmetry? Is there a metric I could/should be using to gauge whether I should be using it?
thanks!
Classify without alignments after your C2 refinement - if there are symmetry-breaking features you should be able to see them by this approach (have tried this for a lot of cases even with quite subtle pseudosymmetry).
I reccommed not totally relying on just a resolution improvement but instead the quality/interpretability of your map is the best way to judge. If you see features appear in your map such as domain shape and/or secondary structure then it justifies the use of applying C2 symmetry.
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Yes 100% agreed with that!
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@Glenn-Masson how many particles this to get to 8A ? If the average CTF of the micrographs is high (5-6A) and also somewhat high average defocus - your data set could be limited there.
That is good all 3D classes have the volume of a dimer.
In addition to the excellent suggestions from @olibclarke and @TMcCorvie, I would process the best I can in C1, explore other tools (e.g. https://phenix-online.org/version_docs/1.20.1-4487/overviews/cryo-em-map-symmetry.html to determine symmetry as well.
With the best (visually complete map) in C1 you can use C2 symmetry relaxation https://guide.cryosparc.com/processing-data/tutorials-and-case-studies/tutorial-symmetry-relaxation in homogeneous of NU-refine. That particle stack can also be symmetry expanded, which will double your particles in C2. Keep in mind if the symmetry group is wrong in refinement you will get unrealistic results.
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Just to clarify - for symmetry relaxation, you want to start from a C2 map (or at least a C1 map that has been aligned such that the pseudo-C2 axis is on Z)
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Thanks for the replies, and I glad to know that I am I’m not doing something too divergent from consensus.
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@Glenn-Masson maybe not your system, but an example of C2 symmetry expansion https://www.ebi.ac.uk/emdb/EMD-27818 the EMD has some examples. They did NU refine in C2 => symmetry expand the particles => local refine with the symmetry expanded particles. This could make the map more interpretable, but I found the impact of symmetry expansion is more obvious with higher symmetry than C2. I also had a case were I processed a nucleosome complex in C2 (because saw it in a paper), but it turned out applying symmetry averaged out a small protein that was bound - so things were better in C1. Good luck in your system.