UC #3 : CryoEM Proteasome

Solve the structure of the proteasome!

Learn how to easily repeat a CryoSPARC refinement in RELION.

Get access to the necessary Job Templates here:

https://app.dectris.cloud/sections/user/share-links/7ZDmN_J98cZUK6bhZQcF6

Extended License Information

Click on your initials in the top right and go to "My Licenses" > "ACTIVATE LICENSE".

You will be prompted to add the following details:

Licence ID: LCID-IUCR-WORK

Activation code: 62411364

Step-by-step

1. Create a new Project
2. 1. Go to Projects
   2. Create New Project
   3. Choose Region US
2. Run CryoSPARC to RELION conversion
3. 1. Go To Library > My Templates
   2. Find the “CryoSPARC to RELION conversion” Job
   3. Select and click “Run Job” on the bottom right
   4. For “cryosparc_project_dir” enter Project “IUCr Workshop : Group A/B/C/D” by double-clicking and navigate to the CryoSPARC project dir: processed/CS-automated-workflow
   5. For “output_parent_dir” enter your newly created Project and select the "processed" subfolder
   6. Click CONTINUE
   7. Check “Show advanced”
   8. Set “DC_COPY_IMAGE_DATA” to True
   9. Click CONTINUE
   10. Stick to the defaults and click RUN JOB
   11. Wait for the Job to finish (~8 min)
3. Run Relion Refine3D
4. 1. Go To Library > My Templates
   2. Find the “Run RELION Job” Job
   3. Select and click “Run Job” on the bottom right
   4. Browse to and select the RELION project dir at “<your new project>/processed/relion_from_cryosparc”
   5. Click CONTINUE
   6. Enter this as “DC_RELION_COMMAND”:
   7. relion_refine_mpi --o Refine3D/job011/run --auto_refine --i Refine3D/job010/run_data.star --ref Refine3D/job010/run_class001.mrc --firstiter_cc --trust_ref_size --ini_high 60 --dont_combine_weights_via_disc --scratch_dir /scratch --ctf --particle_diameter 190 --healpix_order 4 --sym D7 --j 5 --pipeline_control Refine3D/job011/
   8. Click CONTINUE and click RUN JOB
4. Share the Job
5. 1. After the Job is finished (~30 min), check if CryoSPARC’s resolution was reproducible by RELION
   2. Why did the resolution differ from the CryoSPARC result?
   3. Share the link on social media and tag us!
5. Optional: Refine the Refine3D parameters to see if you can get a better resolution!

References:

Campbell, M. G., Veesler, D., Cheng, A., Potter, C. S., & Carragher, B. (2015). 2.8 Å resolution reconstruction of the Thermoplasma acidophilum 20S proteasome using cryo-electron microscopy. eLife, 4. https://doi.org/10.7554/elife.06380

Punjani, A., Rubinstein, J. L., Fleet, D. J., & Brubaker, M. A. (2017). cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nature Methods, 14, 290–296. https://doi.org/10.1038/nmeth.4169

Scheres, S. H. W. (2012). RELION: Implementation of a Bayesian approach to cryo-EM structure determination. Journal of Structural Biology, 180, 519–530. https://doi.org/10.1016/j.jsb.2012.09.006

Asarnow, D., Palovcak, E., Cheng, Y. UCSF pyem v0.5. Zenodo https://doi.org/10.5281/zenodo.3576630 (2019).