3D Classification require particles.alignments3D

Hi everyone,

I am trying to use the 3D Classification job for my extracted particles and using a volume extracted from ChimeraX as an mrc file. The import of the map to CryoSparc didn’t have any problems, but when I tried running the 3D classification job with 2 inputs, (1) exported particles from 2D class and (2) the imported volume as initial volume, I encoutered the following error:

Traceback (most recent call last):
  File "cryosparc_master/cryosparc_compute/run.py", line 60, in cryosparc_compute.run.main
  File "/gpfs/nisbfs/home/cryosparcuser_dgx/cryosparc/cryosparc_worker/cryosparc_compute/jobs/runcommon.py", line 1010, in check_default_inputs
    assert False, 'Non-optional inputs from the following input groups and their slots are not connected: ' + missing_inputs + '. Please connect all required inputs.'
AssertionError: Non-optional inputs from the following input groups and their slots are not connected: particles.alignments3D. Please connect all required inputs.

I thought that the job would not require any alignments to begin? If it does, then should I run an ab-initio job which has the alignment, then merge that together with the imported map? If yes, then how can I do this?

Thank you very much for your help!

It does require alignments - it is classification without alignments (meaning your particles already need to be aligned to some kind of consensus refinement). I would recommend running a homogeneous or non uniform refinement first.

hi @olibclarke, Thank you for your suggestion! I did find that the homogenous refinement does do alignments.3D for the particles and export the particles with that.
And using it the 3D classification is running!

Hi! sorry for intrusion. But how do you do alignments3D? In align 3D maps it kinda does it, but needs couple of volumes?

I have unbanned particles and the resolution dropped significantly when I used them for reconstruction of the volume I got with binned particles. I think due to noise and misalignment…

Hi there,

I don’t think you can use unbinned and binned particles together? MIght be a better idea to re-extract binned particles to unbinned size and try again. Because there will be differences in size and resolution, the reconstruction won’t be good.

I am not sure how to do alignment only, but all Refinement jobs I think have 3D alignments compounded into them.

All the best!