We are just starting to test out 3D-Flex, very exciting!
A couple of queries:
Is 3D-Flex compatible with prior local refinement? E.g. can I take the output of a local refinement, and look at residual flexibility/heterogeneity in the domain afterwards using 3D-Flex?
What is the recommended way to handle symmetric systems? Symmetry expansion and local refinement? Or refine with higher symmetry, but then treat in C1 for the purposes of 3D-Flex?
Cheers
Oli
EDIT:
So far seems to be working with symmetry expanded particles (and improved resolution/detail after flex-reconstruct vs C1); Flex-refine jobs after local refinement have so far only yielded latent coordinates that seem to correspond to rigid rotations of the entire masked region. Perhaps parameter tweaking is required.
Relatedly - how does Flex Data Prep handle data that has been treated with Volume Alignment Tools? In this case, the center of the local reconstruction is not at the center of the particle image. Does Flex data prep crop around the sub-particle (as would happen if one re-extracted with re-centering) or around the center of the particle image? I think probably the latter, but presume that means 3D-flex is not compatible with data treated in this way?
I can comment on your second question: 3D Flex Prep does the latter – it doesn’t crop around the sub-particle, it takes the particle images as they are on disk and crops around the center of the images, then downsamples. For treating data that has been aligned to a sub-particle via Volume Alignment Tools, you could re-extract particles with re-centering enabled, and with a large enough box size to ensure that none of the original protein is “cut off” if any of the particles. (This way the center of the re-extracted images would coincide with the center of the sub-particles.) Note that since the sub-particle is likely not centered at the COM of the protein, this means that slightly larger box sizes are probably needed to do this.
Hi @olibclarke,
How about your result of 3D-Flex following symmetry expansion and local refinement? I face the same situation. Could you please share any ideas? Thanks!
We have no additional updates, but the current workaround to use 3D Flex on arbitrarily re-centered particles would be to use Volume Alignment Tools followed by Re-extraction. We have noted to make 3D Flex work on particles that have been re-centered in Volume Alignment Tools but not re-extracted.
One possible workaround I can think of - the Downsample tool now supports cropping around an arbitrary center (instead of just the box center), so I goess one could go Downsample (cropping around subparticle center → 3D Flex Data prep → 3D Flex Train?
I believe this would also work as well! The main caveat being that if the shift induced by re-centering is significant, downsample accounts for the edges of the images that get shifted out of the FOV by wrapping around, so for large changes to the shift (relative to the box size), I’d likely still recommend re-extracting if possible.
Ah yes – the re-centering in downsample is done in the same way as how shifts are applied to particle images during orientation search (which for computational reasons is via using the Fourier Shift Theorem, which has the wrap-around effect).
But perhaps we could reconsider – would adding noise make more sense? Perhaps it would be important to match the power of any added noise to the power of the particle images?
I guess if the box is big enough it’s fine, but if the box is tight one could run into trouble, having protein features reappearing on the other side of the box - not sure what the best answer is.
I guess it is a pretty niche situation at the end of the day so maybe it isn’t worth worrying about - it might be worth adding a tooltip indicating that this is what is going on though, as the current tooltip is a bit confusing:
Ah I see the confusion – in the case that you want to re-center and expand (pad) the real-space particles (via setting the crop parameter to something larger than the box size), the particles will be mean-padded and then shifted. I’ll make a note to clarify in the re-center parameter option that re-centering is done via the wrap-around method.
