3D variability analysis query

Hi, I’m trying to get a structure of 170kda protein an oligomer that shows

two different conformations open and closed during 2D classification as shown below. After doing abinitio into 4 classes followed by heterogeneous refinement I was able to separate two junk classes and got two conformations of protein after non-uniform and local refinement with closed at 4.3 and open at 6.3A. This was done using 60K particles in each conformation. However, when I do a 3dVA analysis on the closed conformation I see the particles separating into two different clusters and three components but using the 3DVA simple mode I could not play the movies in the Chimera. Can someone advise how to go about this?

Let me correct that 3DVA was done using 120K particles in each conformation as the particles seem to have C2 symmetry which was doubled by symmetry expansion and the number increased to 120K

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Simple mode generates ‘volume series’ which contain the classes arranged from one extreme to the other. In chimera use volume series tool, then use open series and load the maps and hit play. I would rather suggest to take all particles that aren’t “junk”, refine C1, and run 3DVA with 3 components. Then 3DVA display run 2 jobs, one in cluster one in simple. Cluster will give you structures of the many conformations in your heterogeneous dataset (reconstructions and particles - need to perform further refinement of single or recombined classes for high resolution), whereas simple gives only volumes arranged in series