C2 symmetry with symmetry breaking features

3D Variability Analysis
#1

Hi Everyone,

I am new to the structural world but taken some steps toward solving my first CryoEM structure. My protein is ~170KDa. It is a disulphide linked Dimer that exhibit C2 symmetry. The core of the protein is rigid, however the two domains at the periphery are moving independent of each other. I have been trying and experimenting with masks, 3DVA, and C1 vs C2 symmetry in CS and some 3D focused classification on relion but not getting there yet. Any thoughts about how to get the full details of those periphery domains. In the attached image, you can see i might be getting a glance at what could be there or a cloud that lack details

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#2

Hi Bassem, the map looks very promising and like you are on the right track. See the discussion here for some recommendations on how to assess pseudosymmetry in cryosparc.

At first glance, I would try symmetry expansion, followed by local refinement with a mask on the peripheral domain (or single protomer), followed by 3D classification without alignments/shifts in relion using a similar peripheral domain mask. Does 3DVA demonstrate large motions on the periphery and inform on where to place a mask and fulcrum for local refinement?

#3

Hi @user123, all those strategies did not seems to work. I believe owing to the size of the molecule. i am trying now to collect particles de novo in relion. Not sure yet how that will pan out.

#4

I had a similar problem for three of my reconstructions in this manuscript.
https://www.nature.com/articles/s41586-021-03680-3

Symmetry relaxation in Relion was useful to get better results for pseudo symmetry for one of the reconstruction and symmetry expansion + 3d classification without alignment for another. However, your reconstructions should be in correct symmetry axis otherwise it wont work. Basically when you open the map on chimera you should be looking through the sym axis. We requested cryosparc to implement symmetry relaxation, it may be helpful to endorse it so cryosparc scientist may prioritize it.

Good luck.

Alpay

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#5

Hi @alburse,

can you give me more insights into symmetry relaxation and the parameters you used to populate relion to do?

thank you!

#6

You need to align your reference at C2 symmetry axis. Then do a refinement on Relion with C2 (you can also try C1) using usal global alignment settings. Then you need run a local refinement with C1 and use relax symmetry option C2 with global and local search angles to be set to same value like 1.8 (that is how you relion runs a local refinement). You need one of the newer versions of Relion3.1 or Relion4.

#7

My group introduced the symmetry relaxation feature to RELION. I agree, it would be great to have a similar option in CryoSPARC! We believe that this feature is very useful when a complex has pseudo symmetry. Note that relaxing symmetry is different from “symmetry expansion + focussed classification” approach. Both have their own use cases. I would be happy to discuss this further with the cryoSPARC team.

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