I am trying to sort out polarity issues with a protein bound to my filament. I have a polar filament with a binder that interacts in such a way that it should always point towards the same end of the filament. However, I suspect the filament particles are being incorporated into the reconstruction in both directions equally, which is a problem. I have made a tight mask around the filament and did a helical refinement, with the hope that everything will align in the correct orientation. I then expand the helical symmetry and do a 3D classification without alignment (and without a mask) to see which direction the binder protein is pointing. Surprisingly, it is clearly pointing in both orientations equally. To resolve this I use the volume alignment tools to manually flip around the particles for those volumes which point in the other direction (for “3d rotation Euler angles” field I use a value of 3.14, such that the filament is flipped 180 degrees), combine the particles, and do a refinement of the filament + binder. Hoping this will improve the map.
I am just wondering about the step where I do symmetry expansion of the helical parameters, is that necessary? I am having a hard time conceptualizing what symmetry expansion will do in the case of helical symmetry since, unlike point group symmetry, the resulting particle stack has the same number of particles as the input stack.
thanks in advance!