I have a consensus refinement of a glycoprotein to 4.34A global resolution and used it as input for 3DVA with a lowpass filter of 8A and 3 modes.
The motions I observed are large glycan motions, flexing at a hinge point between two subdomains, and breathing within each subdomain. I find this very interesting and would really like to observe it by reconstructing the individual states using NU-refine. Since it is continuous, 3DVA clustering doesn’t seem to be an option for creating particle subsets for downstream refinement. I have seen this post ( Continuous vs Discrete motion in 3DVA - 3D Variability Analysis - cryoSPARC Discuss) but am still not clear on how I can further analyze the motions I observe. Would local refinement with a mask around each subdomain be a good idea? Or could ab initio and hetero refine be used to separate these differences?
Due to the small size (140kDa) and high flexibility, I am not sure if local refinement will be possible after particle subtraction. I would really appreciate any advice!
Output from 3DVA:
component 0 ‘simple’ display movie (purple) and component 2 ‘simple’ display movie (green):
Output from clustering with 5 clusters:
I have since performed NU-refinement on the 5 clusters shown above and obtained maps of around 4-5A for each (there’s still likely heterogeneity due to glycosylation and each cluster is only of around 20-30k particles which would decrease SNR and limit resolution).
When I morph between maps in Chimera I observe the motions shown by 3DVA although a bit less pronounced.
Is this an appropriate way of analyzing my data? Can I now build models into these maps (consensus plus 5 clusters)? The consensus has 120k particles and better features in some regions likely due to higher SNR.