Hello-
I am working with a small particle 60kDa that dimerizes. The particle interacts with a 10kDa protein. Through a standard workflow I captured 50k particles and have a reasonable reconstruction ~3-4 A. I see reasonable density for 90% of the protein- helices,strands and biochemically validated side chain interactions. However, for the 10kDa I see a density that matches the size and its unique shape.
I tried symmetry expansion → 3D classification and I see two classes that have the 10kDa protein and 1 that does not. I used these classes and their symmetry expanded particles for a local refinement but that seems to give a gibberish density.
I did not align the particle prior to symmetry expansion as I do not know how to on ChimeraX, however the 3D classification seemed fine.
Parameters-
3D Classification- Filter Res = 12 , O-EM = 0.6 and Classes = 3
Local Refinement- Default, Local Refine mask prior to symmetry expansion.
Let me know if Ive made any mistakes, if anymore information is required or any tips in general