I would really appreciate some advice.
For my workflow, I used local refinement to gain details for a region of the protein I could not see simply by using homo/heterogeneous refinement. The region of the protein I am performing local refinement on is a C-N terminal domain interface with four chains emanating from the C-terminal domain to eventually coil.
The local refinement worked well but to low resolution. My protein has D4 symmetry so one of my supervisors suggested using symmetry expansion of my particle set in D4 to multiply my dataset and perform subsequent local resolution jobs with C1 symmetry applied to gain resolution.
Although I did gain resolution when doing this, I now have maps with two different paths for the interface. The four chains come from the same place but the direction of the chains differ in the C1 (symmetry expanded) and D4 (non-symmetry expanded) maps.
I have tried subsequent 3D classification with the two maps as inputs to see if I can classify particle subsets that correspond to the different interfaces but this doesn’t yield anything.
I would like some help to see how to determine whether I have true heterogeneity (as in two distinct interfaces), an artefact of the symmetry expansion or something else.
Thank you in advance for your input and advice.