[3dem] Importing Particles from Cryosparc into Relion for Processing

Fri Oct 17 13:59:47 PDT 2025

Hi Dan,

Thank you so much for the response!

I still have some questions that I hope you might be able to answer, help me figure out the best way to do this, or maybe point me in the right direction so I can read some and figure it out.

So just you know what I am trying to do: I have a ~3 A resolution structure in cryosparc. I want to see if the using the Bayesian approach in relion can help me increase the resolution of my structure as well as a couple of other. Since I already have a good set of particles that I picked in cryosparc I want to use those in relion. Someone I am working work with said that the motion correction and CTF estimation is better in relion, so I imported my raw micrographs and ran those jobs to get motion corrected micrographs. I now want to use the particle locations from cryosparc to re-extract those particles from the micrographs i worked up in relion to redo the recontrsuction on.

I have now been able to get the extraction job in relion to work! I first had to import the particles.star file into relion and then I was getting errors about the wrong opticgroup which I then changed and it worked. Though I am not sure if I changed the optics group correctly. haha

Question 1: I am running csparc2star in sbgrid using cs2star. Please let me know how this differs from just running scparc2star. When I tried to run cs2star in sbgrid using some of the commands on your GitHub I got errors since it just wants just the directory path to the cryosparc job folder. It did not let me use the .cs file from exporting from cryosparc which I thought was interesting.

Question 2:  I spent a lot of time changing the _rlnMicrographName in the orignal .star file from the original cryosparc micrograph path to the relion micrograph paths. Is that needed or will relion just ignore that and use just the particle locations from the .star file if I use the relion micrograph as the input for the extraction job? I feel like without those matching relion wouldnt be able to say which micrograph the coordinates belong too but I could be wrong.

Question 3: How do I know if the particle locations are correct? I am now waiting on a 2D classification job to run to see if it outputs the correct looking 2D classes but is there a way to check this easily? I know below you said to use the display option in relion but I don't think I am using it properly since I don't really see anything. I think I am looking at non-corrected images or something because I am not really seeing anything in my images. Side question, can you import .star files into cryosparc or do you need to convert those back to .cs files?

Question 4: What is the best way to change the rlnOpticsGroupName and rlnOpticsGroup? Do I need to change these to match the micrograph.star files from relion?

Question 5:  Do I need to import the .star file into relion like did? And is this where I should change the OpticsGroup or should I do that in .star file before importing it?

Question 6: Do you think it is worth redoing the motion correction and CTF estimation in relion? Someone else I have talked to told me they haven't tried what I am doing yet but instead just uses the extracted particles from cryosparc with micrographs.star file so they haven't ever done what I am trying to do before. I am not wanting to go back to cryosparc or at least right now I don't think i want go back. I know people go back and forth all the time so im not sure what the best way of doing this is.

Sorry for all the questions! I really appreciate the help!  Since I am new to relion and not even sure how to do some simple things in it it's been a struggle. haha. I really want to make sure I am doing this right, so I am not wasting time just running jobs that aren't on particles.

Thanks so much for all your help!!

Lauren

________________________________
From: Daniel Asarnow <dasarnow at gmail.com>
Sent: Thursday, October 16, 2025 8:43 PM
To: Lauren Pellows <Lauren.Pellows at Colorado.EDU>
Cc: 3dem at ncmir.ucsd.edu <3dem at ncmir.ucsd.edu>
Subject: Re: [3dem] Importing Particles from Cryosparc into Relion for Processing

[External email - use caution]

Hi Lauren,
Thanks for using my software. What messages do you see from Relion? You must check both the run.out and run.err files for the extraction job - a major hint is any "particle outside micrograph" warning.

In all our image processing suites but one (nextPYP), particle extraction (using coordinates to duplicate the data around each particle into small images of uniform size) is a required preprocessing step for single-particle analysis jobs. It is up to you whether to import your new micrographs from Relion MotionCorr back into cryoSPARC, or to extract in relion. However, you will notice that the micrographs from Relion are upside-down relative to the ones from cryoSPARC, because Relion flips any .tif file when it is read into memory. Further, Relion and cryoSPARC also use opposite conventions for the Y origin. Further further again, cryoSPARC Import Particle jobs assume the Relion convention in the .star file - the coordinates are inverted. Thus csparc2star.py has several options to account for all use cases.

default : exported coordinates are inverted, so that the same micrographs can be used for extraction in Relion (as mentioned this was the only use case in the first versions of cryoSPARC)
--inverty : exported coordinates are not inverted, so that if they are imported back into cryoSPARC, they will work, e.g. if you only run Class3D in Relion
--flipy : no effect on coordinates, but flips CTF angles to account for the upside-down images (one can always run CTF estimation again instead)

I recommend you always inspect the pick locations on the micrographs either manually, with relion_display, or back in cryoSPARC to avoid wasting time by extracting with wrong coordinates. They can also be verified by running 2D classification again, but it's less efficient.

The same issue will also affect the use of an empirical gain reference produced by relion_estimate_gain. If your data is from a Gatan camera, you will need to use the opposite gain flip (in Y) vs. the gain saved from DM and converted by e2proc2d.py (EMAN2) or dm2mrc (IMOD).

More details, discussion, examples, etc. can be found on the pyem issue tracker and wiki on github.
https://urldefense.com/v3/__https://github.com/asarnow/pyem__;!!Mih3wA!G_FzedcO1gvl7HmT22_wmxhAZA_PRYiuCFd37HO7U41ZBvW3Vi-hYEGa09oXYKhQejRFFJpfk2OAPt5lrm6lRWgvvOLLYHZL$ 

Best,
-da

On Thu, Oct 16, 2025 at 10:55 AM Lauren Pellows via 3dem <3dem at ncmir.ucsd.edu<mailto:3dem at ncmir.ucsd.edu>> wrote:
Hi all,

I am new to cryoEM field and have been working in Cryosparc for all my processing. I am now wanting to use Relion and have been trying to figure out how to import the particles I extracted in cryosparc into relion. I wanted to reprocess my raw micrographs in relion so I imported those ran motion correction and CTF estimation on those. After that I ran csparc2spar on my cryosparc data and now have my particles in a .star file. I also have gone into the particle.star file made sure the _rlnMicrographName in the star file to match the relion processes images. I have now imported the particles.star file into relion but when I run the extraction job I don't get any extracted particles.

I have been reading online and can't find anything conclusive on if I need to run the extraction job. I have read that I dont need run the extraction job since the particles.star file should have all the metadata on the particles, and I can just use the particles.star to run my jobs. I also read that I can rerun the extraction job using the particle coordinates from cryosparc. Since these feel counter intuitive and I keep getting errors I am not sure what to do next. I know that this is a very typically workflow that people do so im hoping this is a pretty straightforward question. Any help or advice anyone has would be a great help!!

Thank you so much,

Lauren Pellows, PhD

Postdoctoral Fellow
Dukovic Lab
University of Colorado, Boulder

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