Issue with Subtomogram Extraction in RELION 5

[ZhichaoJiao]

I am following the subtomogram tutorial in RELION 5 and encountered some issues at the Extract subtomos step:

1. I do not see an Extract subtomos job in the RELION 5 GUI. Instead, I see Make pseudo-subtomos. Has the job name changed? Are they the same?

2. The Make pseudo-subtomos job requires either an Input optimisation set or a combination of particle set, tomogram set, and trajectory. However, the output from the previous Pick tomograms job only contains particles.star. Where can I find the other required files, or how should I generate them?

I am very interested in using RELION 5 for tomographic data processing and would appreciate any guidance.

[bogdantoader]

Hello, which version/commit of Relion are you using? I would recommend using the stable version (branch ver5.0). In any case, Make pseudo-subtomos should be the same as Extract subtomos.

Regarding your second question, jobs such as Extract/Refine3D/Class3D/Bayesian Polishing generate an 'optimistation_set.star' file that points to the a particles.star, tomograms.star and motion.star (the motion.star only after a Polish job has been run). If you don't have the optimsation_set.star file, you can manually input the particles.star file you obtained from the picking job and the tomograms.file from the Tomograms job you used for picking (e.g. Tomograms/job006/tomograms.star). Having said this, if you use the Napari picker in Relion (the 'Pick tomograms' job), there should be an optimisation_set.star file, unless something went wrong? Do you at least see a particles.star file in the Picking job directory?

[ZhichaoJiao]

I checked my RELION version and it is relion-5.0-beta version. Maybe there is a problem with the version. I will upgrade to the latest version and try it.

Regarding the second question, I use the "particles.star" file from [Pick tomograms] and the "tomograms.star" from [Reconstruct tomograms] as the input of [Makepseudo-subtommos] job. I'm not sure if this is correct.

[bogdantoader]

Regardless the name of the job in the version you are using, the extraction should work.
Using the particles.star and tomgorams.star files as you described is correct and the Extract job should work if everything goes well. You can check that you extracted the right particles by running a 'Reconstruct particle' using the output from your Extract job (e.g. the optimisation_set.star).