From 3D-VA I’ve identified a conformational transition - let’s say open to closed channel. When I use 3D-VA-display in “intermediates” mode, it looks like the population distribution is roughly Gaussian along the mode (see below), with the most particles in the middle, “intermediate” state, and fewer particles at either end of the transition.
I then take the 10 reconstructions from 3D-VA display and input them to 3D-classification (beta), using the same mask and particle set as I used for 3D-VA. In this case, the population distribution appears bimodal, with both the “end states” being more populated than the middle (see below).
The results of 3D-classification make more sense to me, as I would expect the middle to be a “transitional” state and hence less populated, but I am wondering if there is any assumption underlying 3D-VA that imposes or biases towards such a population distribution?