I’m interested in locating the tail of an icosahedral virus, while it is easily disregarded through averaging even though I am using C1 symmetry. Could anyone provide guidance on how to align the virus structures to find the tail and perform a local refinement?
If it’s got a tail, it’s pseudo-icosahedral.
This can be pretty tough.
Once particles are picked focussing on the pseudo-icosahedral part, and reconstructed with icosahedral symmetry imposed, I’ve had limited success with trying to lock in a symmetry breaking tail, even with symmetry expansion, customising masks, etc. It sort of works, but if I classify there are always some tails in the wrong place.
A couple of options:
Manually pick the tail, with a box large enough to just catch some of the pseudo-icosahedral part. Then autopicking can work, but take care with inspecting picks and cleaning up in 2D. Depending on what the tail is doing, a C1 reconstruction can give some indication of what sort of symmetry you then need to impose further. This can be time consuming as you may need a lot of manual picks.
Symmetry expand, recenter just outside point of symmetry break (tail 10nm or so away from pseudo-icosahedral capsid with a fairly small box, depending on size), mask out the capsid and classify until all particles not containing that tail view are gone (can be quicker to do manually if you don’t mind moving out of CryoSPARC)**. I found that the pseudo-C5 imposed from the symmetry expansion caused me problems with trying to identify the helical symmetry of the tail later, though.
**Can either be done by visual inspection of the stack, or unpicking the tails, then using
grep to find the list of particles you removed from the symmetry expanded set. It depends how hands on you want to get.
force reference bias, performing het refine with a large obvious tail. then you can reextract, partial signal subtract, 3D class for tail positions etc.
just a suggestion, zero experience