Dear Cryosparc community,
I have an interesting problem arising from hetergeous refinement.
It’s membrane protein, with extracellular bits.
I have a datset contaning particles belong to two conformation. The two conformation’s top view is very different but the side views is very similar to each other, some side view viewing directions have a clear difference.
I suffer from some orentation bias with limited number of side view particles, but enough to give a anisotropic map.
Depending on which intitial model I provide, I get the many side views particles assigned to different conformations in heterogenous refinment.
If I input conforamtion 2x A map as intial, it seperates conformation A and B, and A conformation has most side views. Non uniform refines to 3A
If I input conforamtion 2x B as intial, it seperates conformation A and B, and B conformation has most side views. Non uniform refines to 3.2A.
If I input conformation 1xA 1xB as intial, it sepetares A and B, and A conformation has most side views.Non uniform refines to 3A.
Maps are checked and verified to have expected features in correct location.
When I checked the two sets for duplicated particles. 2D class reviews duplicated particles are all side views.
Low pass filter is turned on at 20A.
I ran a 2D classification on set A, it’s clear that some side views clearly belongs to conformation B, but misassgiend to set A.
So, my question is, what is the algorithm behind hetergenous refinment, and why would it be quite heavily influenced by the intial model?
Unfortunately Due to NDA reason, I can’t share details of the proteins or the images, but would be happy to describe as much as I could.