Heterogeneous refinement to separate viral pore mutants?

Hi all,

I am currently processing a dataset of a mixture of 3 different pore mutants of the same viral capsid.
The mutations around the pores should change the pore structure/size noticeably. What would be the
best strategy to separate the particles of the three mutants? I was thinking of doing Heterogeneous
Refinement with 3 input volumes. However, the first round resulted in most (98%ish) particles going
into one class. Would it be a good idea to do another round of Heterogeneous Refinement using only
the 98% class? I am fairly confident that this class contains all good particles of all 3 mutants because
it’s resolution is surprisingly bad compared to what I would expect for a non-mixed sample of the same
quality. How many input volumes should I use for the second round? Also, would I have been able to
separate the mutants in the first round if I had added say 6 volumes?

Thanks for your help!