Hey Meranda,
Yeah, that’s really small – that in combination with the fact that you aren’t using subtracted particles (and still are seeing chunks of volume gone) leads me to suspect that alignments are definitely going awry. Certainly try to see if you can make a mask covering more of the protein (ideally, the largest part of the protein that’s suspected to move as a rigid body, and that preserves tertiary structure). For the fulcrum, try setting it to the center of mass of the mask. Unfortunately there isn’t currently a way to do this in cryoSPARC automatically – you’ll have to use Chimera’s measure center command (see this post for some details from DanielAsarnow – also a generally useful thread for tips in using local refinement).
Best, Michael