Hi @GhonchehM this is an interesting question. In fact in cryoSPARC to make things simple, we don’t even use the symmetry constraint during alignment of particles. Typically symmetry reduces the search space (as you see in Relion) but in cryoSPARC alignment using the Branch-and-Bound algorithm is very fast, so that we don’t attempt to exploit this gain, to keep the code simpler. Symmetry is only used during reconstruction (backprojection) in the typical way (same as Relion) to incorporate information from each particle multiple times (the order of the symmetry). This is why in cryoSPARC, particle alignments are spread evenly in all asymmetric units, rather than being confined to a single one as in Relion.