I am trying to spearate subparticles of virus capsid with and w/o Fab attached - a classic case of compositional heterogeneity. Ideally, after running 3D classifcation one should have two classes, 1 with Fabs bound and another without. I am relatively inexperienced with some CryoSparc’s 3D classification features and it is not clear how many of the different options in the job builder affect the outcome. Principally, I have been playing with the following params -
class3D_online_em_lr_init
class3D_class_anneal_beta
class3D_init_mode (simple, PCA, init vols)
Additionally, I have tried with particles stacks with capsid density subtracted from the projections (which is the mathematically right way of dealing with this situation). But, I am still struggling to get clean separation.
Are there any parameters I should be taking into account to force the classes apart? (I saw a parameter called “force hard separation”, but I am not sure if that is the right option for the job.)
Ideally, one would touch the different parameters to see what works, but given all the options, I could be stuck with twiddling knobs until the end of days. So this is why I am turning to the collective wisdom accrued within the CS community.
Many thanks in advance for your suggestions.
Best,
Pranav