Overview

CryoSPARC and Relion are both useful tools for analyzing cryoEM data. General consensus in the field is that the most powerful analyses use a combination of both. Outlined below is a tutorial for taking a particle stack from cryoSPARC to Relion 5 on Montana State University's HPC cluster, Tempest.

The general flow looks something like this (adapted from DOI: 10.3791/63387) and uses a script (c2relion_helper_script.sh) customized for Tempest (as of 2023-11-13, only available to Wiedenheft lab members).

cryosparc to relion

Getting started

  • Command line access to Tempest with X11 forwarding enabled
    • Make sure you are using the -X flag when using ssh
    • ssh -X <yournetID>@tempest-login.msu.montana.edu
  • You will need the following:
    • A relion directory inside of your project directory
      1. Wiedenheft lab projects are organized so that each project directory has 
        1. Raw data micrographs
        2. A cryoSPARC directory
        3. A relion directory 
          • To check if a Relion directory exists, use the following commands:
          • cd /mnt/blackmore/mcb-wiedenheft/EM-data/<path_to_your_project>
          • ls /mnt/blackmore/mcb-wiedenheft/EM-data/<path_to_your_project>
      1. If a relion directory doesn't exist, use the following commands  (replacing <path_to_your_project> with the path to your project directory
      2. mkdir /mnt/blackmore/mcb-wiedenheft/EM-data/<path_to_your_project>/relion

        cd /mnt/blackmore/mcb-wiedenheft/EM-data/<path_to_your_project>/relion

project directoriesYour project directory should now look like this (letters indicate directory as outlined above, box around relion directory).

    • A homogenous or non-uniform refinement job from cryoSPARC (with corresponding JXXX job number)
      • Take note of the final iteration number:
      • iteration number
    • A cryoSPARC extraction job that contains your particles of interest.
      • If you used a multi-threaded extraction job initially, you will need to re-extract your particles used in the refinement as a single extraction job.
      • To reduce memory and cache requirements, we recommend doing this regardless of your initial extraction strategy.

Running c2relion_helper_script

  • Once you have completed the above steps, you are ready to import your particles into Relion.
  • Load the relion module using the following command:
    • module load cryoem/relion/5.0
  • Add the location of the c2relion_helper_script to your PATH variable
    • export PATH=$PATH:/home/group/blakewiedenheft/software/bin
  • Run the c2relion_helper_script and follow the prompts in the terminal.
    • c2relion_helper_script.sh
    • Note: do not include the trailing slash in your relion and cryosparc paths!
    • Note when using projects from old cryoSPARC versions imported into updated cryoSPARC:  You may get a "File Not Found Error" when converting the particles.cs to particles.star. In case you get this error, navigate into the appropriate job directory and copy the particles.cs and particles_passthrough.cs files to new versions that have the appropriate prefix.
      • For example, P5 J402 was a Refinement conducted in cryoSPARC 3. The prefixes are:
        • cryosparc_P5_J402_007_particles.cs 
        • P5_J402_passthrough_particles.cs
      • These need to be copied, and the copies should be renamed to:
        • J402_007_particles.cs
        •  J402_passthrough_particles.cs
  • This script will create a user-specific relion project directory. Follow the prompts from the script, and cd into that directory.
    • It is CRITICAL that you cd into this directory before launching relion to avoid conflicting analyses between users - Relion is NOT as user friendly as cryoSPARC.

Launching Relion

  • From your user-specific relion project directory, launch relion with:
    • relion & 
  • You will now need to import your particle stack into Relion using the Import job.
    • In the Movies/mics tab, set "Import raw movies/micrographs" to "No"

import-movies-tab 

      • In the Others tab, set your input file to the .star file in your user-specific relion project directory. Set Node type to "Particles STAR file (.star)".

import-other-tab

  • Click the "Run" button (don't worry about anything on the "Running tab")

3D Classification in Relion

Builld initial models in Relion

  • In the "Input" tab, set your particle.star file to the imported .star file (Import/jobXXX/<particles_file_name.star>)
    • initial-volume-io-tab
  • In the "Compute" tab, change the followig fields:
    • Number of pooled particles: 30
    • Use GPU acceleration? Yes
    • Defaults are fine for the rest.
    • initial-model-compute-tab
  • In the "Running" tab, change the following fields:
    • Number of MPI procs: 1
    • Number of threads :12
    • Queue name: gpuunsafe (generally) or gpupriority (rarely)
    • Tempest account: group-blakewiedenheft (generally) or priority-blakewiedenheft (rarely)
    • # of GPUs: 1
    • Defaults are fine for the rest.
    • initial-model-running-tab

3D classification

3D classification is a powerful for sorting out particle heterogeneity once you have a relatively clean particle stack.