Hello,
I have a complex with a D-type symmetry, and using symmetry expansion, 3D classification, and local refinement with masking, I have a class of asymmetric subunits that have a substrate bound (others do not have substrates). I would like to map these substrate-bound subunits back to the entire complex to see where the substrate-bound asymmetric subunits are in the entire protein complex.
Does anyone have suggestions or know how to do this on Cryosparc (or elsewhere)?
Since you carried out symmetry expansion, it’s likely that all sites are partially occupied. If conformation of the whole complex shifts when one site is bound, that may impair/stop binding at other sites in the complex, but saying “this site is bound on this complex symmetry subunit” may prove difficult.
You could try using the one site locally refined map as a reference in C1 symmetry for a heterogeneous refinement, but unless there is a dramatic conformational change upon binding the protein signal would still dominate alignments (particularly early on) and make any resulting map a “best guesstimate” even with the most generous interpretation, and at worst a misleading fantasy.
Thank you for your reply. May I ask for additional clarifications? I realized that I may have been unclear.
After symmetry expansion, I did local refinements and 3D classifications of a single asymmetric subunit.
I forgot to add that with the substrate bound, there’s a ~200 amino acid domain that is resolved (~ 3.5 Å).
3D job splits the asymmetric subunits based on the substrate bound (with a resolved 200 aa domain) and those without the resolved 200 aa domain (~1/3 of the total asymmetric subunits).
So at this point, I have a class of asymmetric subunits that I know is different from the rest (has resolved 200 aa domain, and most likely substrate)–unless there is a flaw in the workflow that I have not caught.
All I want to do is to “map back” a selection of these asymmetric subunits back to the entire D-type complex to see if there’s a pattern.
Is this doable, or would doing this still be an over-interpretation even with the 200 aa domain differences?
Also, is there a reason why a heterogeneous refinement be preferred over 3D classification, especially if the local changes are small compared to the entire complex? The only difference between the asymmetric subunits that would be observed at the resolution I am working at is the presence/absence of a 200 aa domain.
Any clarification would be much appreciated, thank you!