I am trying to revive this topic. My particles in Cryosparc through Patch Motion > Patch CTF > … > Refinement.
I can convert everything well according to the guideline from pyem using Methods 2. Extracted the particles and did a test reconstruction, and everything looked good. So particle coordinates & orientations are well converted by pyem. But when I polished it, the output particles were not at the right location at all. Does anyone see anything similar?
Extracted the particles from the cryoSPARC micrographs, right? You have probably needed to invert the coordinates and flip the CTF values.
Hi Daniel,
So apparently, to polish, I need to invertY and flipY (flip CTF as you said). But to test reconstruction by extract from the coordinate and reconstruct, I would use not InvertY (with --inverty option). It is a bit confusing but it works.
In this case it’s because in your “new” extraction test the particles are coming from the same drift-corrected micrographs you’ve had all along in cryoSPARC. Then only when you run polishing are you getting something based on Relion looking into the movies directly.
If you would instead run motion correction from scratch in Relion, then those new drift-corrected micrographs in Relion would have the same conventions as the Relion polishing job. (And in that case, extracting & refining would really tell you if Relion is getting the right coordinates. It would also let you use definitely correct CTF and alignment parameters, instead of the ones converted by me from whatever was estimated in cryoSPARC, based on flipping the images. Finally, Relion would also be able to start the polishing trajectories from the local patch trajectories found during initial motion correction. For that reason this is my recommended approach).
I’m running into the same issue as other users (“There is no movie metadata STAR file for any micrographs!”) – apparently, in a bid to consolidate data and save storage space I deleted the files for the initial “Import Movies” job, so while I have motion-corrected .mrc files from the subsequent “Motion Correction” job, the corrected_particles.star file points to un-corrected .tif files from the Import job that do not exist anymore.
When I re-run or clone the import job, the generated .tif files have different prefixes (file names formatted as {prefix}_moviename.tif) so the file names are not identical to the previous files. Is there any way to remedy this issue beyond full reprocessing from the initial data import?
@forrest If the problem is that the path to the tif files has changed and you did not delete or otherwise modify the original Import Movies job, you may be able to update the targets of the
symbolic links inside that original Import Movies job’s imported/ subdirectory (guide).
Hello Everyone,
I apologize for reopening the this thread again but I had a question in similar lines to this discussion. So if one polishes their particles and then re-extracts the polished particles in a binned box ( 2 times binning ) to further clean the data, does one then extracts the polished particles or the unpolished particles?
If they are the unpolished particles, is it possible to get the information that was stored during polishing into the newly cleaned dataset (to improve the resolution) without actually doing polishing again?
Hi,
Re-extraction sources the averaged micrographs, so these will be equivalent to unpolished particles. You can also simply downsample the polished particles for curation instead to get to a similar place. Alternatively, just use the polished particles as is, provided memory footprint isn’t too onerous?
In the case of re-extraction/downsampling, after identifying and discarding suboptimal particle images, you can perform an intersect routine (Particle Sets Tools) to fish out the particles of interest from the original polished set.
Cheers,
Yang
Hello Yang,
Thanks for clearing my doubt regarding this.
I will try this out.
Thanks a lot for the help.
To supplement @leetleyang’s correct answer, just think of polishing as “fancy extraction” - if you want new fancy particles (e.g. at a different box) you will always have to re-run it. Unfortunately there is no feature to re-use trajectory information to make it faster the second time.
Off on a bit of a tangent, if what you wish to do is simply repeat a polish job but with different box/resampling parameters, there is a hack-y way of reusing previous trajectories/B-factors and skip ahead to the frame recombination step:
CCPEM: Relion 3.1 – re-extracting polished particles from movies
Last did it in RELION-4, but I imagine it should work with RELION-5 as well.
Cheers,
Yang
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Okay thanks a lot, I will give it a shot.