I’m working with a 240 kDa, homohexamer that displays 3D conformational change in 3DVA with a 20 A high pass filter. Analysis of data sets with the apo and ligand bound structures show that there is more variability in the apo data set, as you might expect.
The issue is that this is not reflected in the refined maps. Both the ligand bound and apo maps overlap and show both in a closed conformation after localized refinement where I am running symmetry expansion and centering on a subunit by placing the fulcrum point at the center of my mask.
Is this because the protein exists in the closed conformation most often, even in the apo data set? Or is it because localized refinement is aligning the rest of the protein to the area selected for refinement refinement. Is there a way to validate 3D variability in the apo data set?