Import cryoSPARC particles back to cryoSPARC after relion processing

Nitesh · May 25, 2022, 3:22am

Hi ,
I am a new user to cryoSPARC.
I have pre processed my cryoem SPA data in to cryoSPARC and performed 2D and 3 D classification in cryoSPARC. After that I use "csparc2star.py "command to make particle stack file for relion 4. I have performed 2 rounds of the 3D classification in relion 4. Now I have run select job in Relion to select particle belong to particular class. I wanted to performed non-uniform refinement on these selected particle and so I tried to import them back to cryoSPARC. I am getting following error while I am trying to import the particles in to cryoSPARC.

[CPU: 724.8 MB] Traceback (most recent call last):
** File “cryosparc_master/cryosparc_compute/run.py”, line 85, in cryosparc_compute.run.main**
** File “/data/cryosparc_user/applications/cryosparc_master/cryosparc_compute/jobs/imports/run.py”, line 141, in run_import_particles**
** assert False, “Files referenced by the input star file are missing.”**
AssertionError: Files referenced by the input star file are missing.

In earlier discussion on cryoSPARC discussion platform I have read that people suggested that one should run particle extract job in relion first and then should use extract job particles directly to import in cryoSPARC. I wanted to extract these particles in relion. For the same, I don’t know what I should give as input in" micrographs.star" file option in relion for particle extraction. How I can use my preprocessed micrographs file (performed in cryoSPARC) in relion to extract these selected particles.

Could some one explain point wise what I should do?

Thank you
Nitesh

Lai · May 25, 2022, 3:42am

Hi,
Did you make a soft link when you imported particles into relion4? Could you show the first 60 lines of your star file ?

Nitesh · May 25, 2022, 9:39pm

Hi Lai,
I have not made soft link during importing my file.
I prepared .star file using “Csparc2star.py”.
Following are the top 60 lines from my star file

data_optics

version 30001

data_optics

loop_
_rlnOpticsGroupName #1
_rlnVoltage #2
_rlnImagePixelSize #3
_rlnSphericalAberration #4
_rlnAmplitudeContrast #5
_rlnOpticsGroup #6
_rlnImageSize #7
_rlnImageDimensionality #8
opticsGroup3 300.000000 1.069400 2.700000 0.070000 1 256 2

version 30001

data_particles

loop_
_rlnImageName #1
_rlnMicrographName #2
_rlnCoordinateX #3
_rlnCoordinateY #4
_rlnAngleRot #5
_rlnAngleTilt #6
_rlnAnglePsi #7
_rlnOriginXAngst #8
_rlnOriginYAngst #9
_rlnDefocusU #10
_rlnDefocusV #11
_rlnDefocusAngle #12
_rlnPhaseShift #13
_rlnCtfBfactor #14
_rlnOpticsGroup #15
_rlnRandomSubset #16
_rlnClassNumber #17
000001@J103/extract/000002771751821158702_stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted_particles.mrcs J13/motioncorrected/stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted.mrc 177.000000 4550.000000 -65.19201 13.239437 70.236809 -1.08611 -1.17300 14683.398438 14344.605469 14.751963 0.000000 0.000000 1 2 1
000002@J103/extract/000002771751821158702_stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted_particles.mrcs J13/motioncorrected/stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted.mrc 2739.000000 5415.000000 54.741737 108.951607 -154.68651 0.217222 0.043444 14683.398438 14344.605469 14.751963 0.000000 0.000000 1 1 1
000003@J103/extract/000002771751821158702_stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted_particles.mrcs J13/motioncorrected/stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted.mrc 332.000000 4969.000000 148.897202 173.219376 -79.84690 0.390999 -0.30411 14683.398438 14344.605469 14.751963 0.000000 0.000000 1 2 1
000004@J103/extract/000002771751821158702_stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted_particles.mrcs J13/motioncorrected/stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted.mrc 3587.000000 4167.000000 -14.92212 64.874825 -45.55165 0.912332 0.043444 14683.398438 14344.605469 14.751963 0.000000 0.000000 1 1 1
000005@J103/extract/000002771751821158702_stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted_particles.mrcs J13/motioncorrected/stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted.mrc 3653.000000 2427.000000 31.039831 162.970001 -80.29691 2.389441 -1.69433 14683.398438 14344.605469 14.751963 0.000000 0.000000 1 2 1
000006@J103/extract/000002771751821158702_stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted_particles.mrcs J13/motioncorrected/stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted.mrc 3436.000000 2172.000000 51.481815 77.088173 -104.30844 0.043444 -0.21722 14683.398438 14344.605469 14.751963 0.000000 0.000000 1 1 1
000007@J103/extract/000002771751821158702_stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted_particles.mrcs J13/motioncorrected/stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted.mrc 3428.000000 1735.000000 -13.47874 101.598862 164.890701 0.422337 -0.56478 14683.398438 14344.605469 14.751963 0.000000 0.000000 1 2 1
000008@J103/extract/000002771751821158702_stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted_particles.mrcs J13/motioncorrected/stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted.mrc 1042.000000 537.000000 161.239960 25.365162 -88.25764 0.651666 -0.47789 14683.398438 14344.605469 14.751963 0.000000 0.000000 1 1 1
000009@J103/extract/000002771751821158702_stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted_particles.mrcs J13/motioncorrected/stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted.mrc 440.000000 1326.000000 82.649467 94.964462 20.719711 -2.82388 -6.06609 14683.398438 14344.605469 14.751963 0.000000 0.000000 1 2 1
000010@J103/extract/000002771751821158702_stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted_particles.mrcs J13/motioncorrected/stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted.mrc 3283.000000 2466.000000 162.273300 80.952202 116.362267 0.564777 0.564777 14683.398438 14344.605469 14.751963 0.000000 0.000000 1 1 1
000011@J103/extract/000002771751821158702_stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted_particles.mrcs J13/motioncorrected/stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted.mrc 3329.000000 3290.000000 139.796021 63.282040 33.394310 6.386323 -1.34678 14683.398438 14344.605469 14.751963 0.000000 0.000000 1 2 1
000012@J103/extract/000002771751821158702_stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted_particles.mrcs J13/motioncorrected/stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted.mrc 742.000000 5340.000000 -87.40295 101.550674 -70.57821 -4.24741 1.847823 14683.398438 14344.605469 14.751963 0.000000 0.000000 1 1 1
000013@J103/extract/000002771751821158702_stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted_particles.mrcs J13/motioncorrected/stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted.mrc 256.000000 2722.000000 54.721489 79.328232 -119.45683 -0.13033 0.304111 14683.398438 14344.605469 14.751963 0.000000 0.000000 1 2 1
000014@J103/extract/000002771751821158702_stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted_particles.mrcs J13/motioncorrected/stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted.mrc 982.000000 279.000000 -13.91599 127.065880 -92.61176 1.086109 0.390999 14683.398438 14344.605469 14.751963 0.000000 0.000000 1 1 1
000015@J103/extract/000002771751821158702_stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted_particles.mrcs J13/motioncorrected/stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted.mrc 199.000000 1497.000000 -10.86641 117.621750 -149.40330 -0.56478 -0.47789 14683.398438 14344.605469 14.751963 0.000000 0.000000 1 2 1
000016@J103/extract/000002771751821158702_stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted_particles.mrcs J13/motioncorrected/stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted.mrc 2748.000000 3114.000000 -165.73912 43.398823 153.706543 0.778941 0.367748 14683.398438 14344.605469 14.751963 0.000000 0.000000 1 1 1
000017@J103/extract/000002771751821158702_stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted_particles.mrcs J13/motioncorrected/stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted.mrc 2926.000000 4935.000000 -23.80281 119.394981 169.342514 1.781219 0.477888 14683.398438 14344.605469 14.751963 0.000000 0.000000 1 2 1
000018@J103/extract/000002771751821158702_stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted_particles.mrcs J13/motioncorrected/stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted.mrc 3508.000000 2750.000000 -19.87011 45.125004 -93.78854 0.372428 0.651666 14683.398438 14344.605469 14.751963 0.000000 0.000000 1 1 1
000019@J103/extract/000002771751821158702_stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted_particles.mrcs J13/motioncorrected/stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted.mrc 684.000000 2191.000000 -34.90185 89.383591 91.633949 0.564777 -0.30411 14683.398438 14344.605469 14.751963 0.000000 0.000000 1 2 1
000020@J103/extract/000002771751821158702_stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted_particles.mrcs J13/motioncorrected/stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted.mrc 473.000000 2633.000000 -44.21791 95.742020 47.142155 -0.91233 -1.60744 14683.398438 14344.605469 14.751963 0.000000 0.000000 1 1 1
000021@J103/extract/000002771751821158702_stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted_particles.mrcs J13/motioncorrected/stack_214-18_001_X+0Y-1-0_patch_aligned_doseweighted.mrc 3996.000000 2948.000000 -91.30264 108.547974 74.389374 0.503077 -0.13033 14683.398438 14344.605469 14.751963 0.000000 0.000000 1 2 1

Best
Nitesh

Lai · May 26, 2022, 12:50am

Hi,
Did you run relion under the cryosparc project directory?
You can make a soft link and try again. Fisrt make a directory named ‘J103/extract’ in you relion directory (with the ‘default_pipeline’ file) and then use ‘ln -s /xx/xx/xx/Pxx/J103/extract/*.mrc ./’ (xx means the path to your cryosparc project). Rename the suffix ‘mrc’ using ‘rename mrc mrcs *.mrc’. And I think you had edited the star file to change ‘mrc’ to ‘mrcs’.(sed -i 's/particles.mrc/particles.mrcs xx.star). Run 3D classification job and import again. Hope it can help.
Best wished.

Nitesh · May 26, 2022, 6:24pm

Hi Lai,
Thankyou for the information.
I will try this.
Best
Nitesh