Hi
I have a data set that consists of an icosahedral capsid bound by an antibody. When I carried out icosahedral reconstruction I found that a surface loop of the capsid had been shifted by the binding of the antibody leading to apparent disorder in the region. I did a local refinement to see if I could capture the new conformation of the loop. As part of the local refinement I did a 3d classification of my sub particles to class out any sub particles that weren’t featuring antibody binding and I was able to capture a class that represents ~20% of my particles and gives me a decent idea of where this loop shifted to. That said, I am hoping to refine this further to see if this is a hard shift to a single other conformation, several possible positions, or a more general flexible position centered around the conformation I was able to find. My current approach is to take this new map with the loop shift use it as a focus mask and repeat iterations of 3d classification to try to further class out different states for this loop. I’m wondering if anyone could tell me if this is a valid approach or if there would be a better approach that I could use? More specifically, I wanted to know if this is the right way to employ a focus mask or if I should be using the new map as an initial volume?
As an aside my initial 3d classification of my sub particles did produce a class without antibody binding that represents ~25% of my subparticles and refinements from this class show the loop in its expected conformation for the capsid.