Hello,
I need assistance with reconstructing maps from specific 3D variability component frames. I attempted to use the 3D variability cluster mode to segregate particles for reconstruction or non-uniform refinement. Unfortunately, none of the clusters provided a satisfactory map as good as the 3D var simple mode component frame. Can you guide me on how to reconstruct a map from the frames of the 3D variability component at the simple mode? Many thanks for the answer.
Best,
Yuhang
not possible (to my knowledge). maps generated with simple mode do not necessarily result from reconstruction of a set of particles, rather from interpolation between two endpoints. this mode is used to describe the motions. you must use cluster mode (or intermediate) to get particle sets that can be used. One trick in your situation is to import the preferred volume from your “simple mode” series and use it as a template for other 3D sorting methods. Be careful though to avoid bias; the preferred volume may not be a true representation of a form the protein (complex) takes, especially if cluster mode fails to observe.
Use the “intermediates” mode, with the rolling window parameter set to -1, selecting only the desired component (with save particle sets on). this will provide reconstructions of equally populated bins of particles along the component axis.