Riboosome 80S has a severe orientation bias

3D Reconstruction
1

Hey all

Is anyone working on a ribosome dataset? I am confused as to why my 80s ribosome has a a very severe orientation bias?

I have obtained 80S ribosome by sucrose cusion or tag affinity purification, no glycerol and no detergent in my sample.

Blob pick can work well with setting min/max diameter = 200/350, followed by an extract job with box size = 600.

select 2D class as followed

J870_selected_14_classes
J870_selected_14_classes1010×206 29.5 KB

But when I running Ab-initio job, it will failed.

  1. Feb 23, 2025, 4:12:00 PMTraceback (most recent call last): File “cryosparc_master/cryosparc_compute/run.py”, line 95, in cryosparc_master.cryosparc_compute.run.main File “cryosparc_master/cryosparc_compute/jobs/abinit/run.py”, line 285, in cryosparc_master.cryosparc_compute.jobs.abinit.run.run_homo_abinit File “/spshared/apps/cryosparc4/cryosparc_worker/cryosparc_compute/noise_model.py”, line 119, in get_noise_estimate assert n.all(n.isfinite(ret)) AssertionError

J871_structure_for_class_000_iteration_800
J871_structure_for_class_000_iteration_8001062×285 124 KB

what should I do with this dataset?

2

@hai Regarding the failed ab initio reconstruction job, please can you post the outputs of these commands

csprojectid=P99 # replace with actual project ID
csjobid=J199 # replace with id of the failed job
cryosparcm cli "get_job('$csprojectid', '$csjobid', 'job_type', 'version', 'instance_information', 'status',  'params_spec', 'errors_run')"
cryosparcm eventlog $csprojectid $csjobid | tail -n 40
3

Occasionally get this error for ab initio usually with many classes.

Error:
Traceback (most recent call last):
File “cryosparc_master/cryosparc_compute/run.py”, line 116, in cryosparc_master.cryosparc_compute.run.main
File “cryosparc_master/cryosparc_compute/jobs/abinit/run.py”, line 301, in cryosparc_master.cryosparc_compute.jobs.abinit.run.run_homo_abinit
File “/home/cryosparc_user/cryosparc/cryosparc_worker/cryosparc_compute/noise_model.py”, line 119, in get_noise_estimate
assert n.all(n.isfinite(ret))
AssertionError

image
image776×376 105 KB

However on earlier iterations has particle looking volumes. Below is one where killed the job at earlier timepoint and marked as complete to run an hetero refine on.

image
image782×386 82.4 KB

4 
(base) [cryosparc_user@localhost ~]$ csprojectid=P17
(base) [cryosparc_user@localhost ~]$ csjobid=J200
(base) [cryosparc_user@localhost ~]$ cryosparcm cli "get_job('$csprojectid', '$csjobid', 'job_type', 'version', 'instance_information', 'status',  'params_spec', 'errors_run')"

{'_id': 'xxx', 'errors_run': [{'message': '', 'warning': False}], 'instance_information': {'CUDA_version': '11.8', 'available_memory': '116.50GB', 'cpu_model': 'Intel(R) Xeon(R) CPU E5-2640 v4 @ 2.40GHz', 'driver_version': '12.4', 'gpu_info': [{'id': 0, 'mem': 11707547648, 'name': 'NVIDIA GeForce GTX 1080 Ti', 'pcie': '0000:02:00'}, {'id': 1, 'mem': 11707547648, 'name': 'NVIDIA GeForce GTX 1080 Ti', 'pcie': '0000:03:00'}, {'id': 2, 'mem': 11707547648, 'name': 'NVIDIA GeForce GTX 1080 Ti', 'pcie': '0000:82:00'}, {'id': 3, 'mem': 11707547648, 'name': 'NVIDIA GeForce GTX 1080 Ti', 'pcie': '0000:83:00'}], 'ofd_hard_limit': 262144, 'ofd_soft_limit': 1024, 'physical_cores': 20, 'platform_architecture': 'x86_64', 'platform_node': 'localhost.localdomain', 'platform_release': '4.18.0-348.7.1.el8_5.x86_64', 'platform_version': '#1 SMP Wed Dec 22 13:25:12 UTC 2021', 'total_memory': '125.52GB', 'used_memory': '7.96GB'}, 'job_type': 'homo_abinit', 'params_spec': {'abinit_K': {'value': 10}, 'compute_use_ssd': {'value': False}}, 'project_uid': 'P17', 'status': 'failed', 'uid': 'J200', 'version': 'v4.6.0'}
(base) [cryosparc_user@localhost ~]$ cryosparcm eventlog $csprojectid $csjobid | tail -n 40

[Sat, 22 Feb 2025 04:52:56 GMT] [CPU RAM used: 4089 MB]    -- Class  1 -- lr: 0.10 eps: 247368961144398997838455705174016.00 step ratio : 0.0000 ESS R:  1.002 S: 11.994 Class Size: 0.0% (Average: 10.1%)
[Sat, 22 Feb 2025 04:52:56 GMT] [CPU RAM used: 4089 MB]    -- Class  2 -- lr: 0.10 eps: 247368961144398997838455705174016.00 step ratio : 0.0000 ESS R:  1.003 S: 11.987 Class Size: 0.0% (Average: 9.7%)
[Sat, 22 Feb 2025 04:52:57 GMT] [CPU RAM used: 4089 MB]    -- Class  3 -- lr: 0.10 eps: 247368961144398997838455705174016.00 step ratio : 0.0000 ESS R:  1.002 S: 11.991 Class Size: 0.0% (Average: 10.3%)
[Sat, 22 Feb 2025 04:52:57 GMT] [CPU RAM used: 4089 MB]    -- Class  4 -- lr: 0.10 eps: 247368961144398997838455705174016.00 step ratio : 0.0000 ESS R:  1.001 S: 11.986 Class Size: 0.0% (Average: 10.1%)
[Sat, 22 Feb 2025 04:52:57 GMT] [CPU RAM used: 4089 MB]    -- Class  5 -- lr: 0.10 eps: 247368961144398997838455705174016.00 step ratio : 0.0000 ESS R:  1.007 S: 11.998 Class Size: 0.0% (Average: 9.8%)
[Sat, 22 Feb 2025 04:52:57 GMT] [CPU RAM used: 4089 MB]    -- Class  6 -- lr: 0.10 eps: 247368961144398997838455705174016.00 step ratio :   nan ESS R:  1.001 S: 12.000 Class Size: 62.1% (Average: 10.1%)
[Sat, 22 Feb 2025 04:52:57 GMT] [CPU RAM used: 4089 MB]    -- Class  7 -- lr: 0.10 eps: 247368961144398997838455705174016.00 step ratio :   nan ESS R:  1.006 S: 12.000 Class Size: 32.7% (Average: 9.7%)
[Sat, 22 Feb 2025 04:52:57 GMT] [CPU RAM used: 4089 MB]    -- Class  8 -- lr: 0.10 eps: 247368961144398997838455705174016.00 step ratio : 0.0000 ESS R:  1.002 S: 11.986 Class Size: 0.0% (Average: 10.1%)
[Sat, 22 Feb 2025 04:52:57 GMT] [CPU RAM used: 4089 MB]    -- Class  9 -- lr: 0.10 eps: 247368961144398997838455705174016.00 step ratio : 0.0000 ESS R:  1.001 S: 11.999 Class Size: 5.3% (Average: 10.1%)
[Sat, 22 Feb 2025 04:53:03 GMT] [CPU RAM used: 4072 MB]   Done iteration 02682 of 03069 in 14.865s. Total time 38061.1s. Est time remaining 5950.4s.
[Sat, 22 Feb 2025 04:53:03 GMT] [CPU RAM used: 4152 MB] ----------- Iteration  2683 (epoch 1.389).  radwn 48.00  resolution 12.00A  minisize  300  beta 0.00
[Sat, 22 Feb 2025 04:53:11 GMT] [CPU RAM used: 4150 MB]    -- Class  0 -- lr: 0.10 eps: 331867415258755320856599144169472.00 step ratio : 0.0000 ESS R:  1.002 S: 11.994 Class Size: 0.0% (Average: 9.9%)
[Sat, 22 Feb 2025 04:53:11 GMT] [CPU RAM used: 4150 MB]    -- Class  1 -- lr: 0.10 eps: 331867415258755320856599144169472.00 step ratio :   nan ESS R:  1.005 S: 12.001 Class Size: 9.0% (Average: 10.1%)
[Sat, 22 Feb 2025 04:53:12 GMT] [CPU RAM used: 4150 MB]    -- Class  2 -- lr: 0.10 eps: 331867415258755320856599144169472.00 step ratio : 0.0000 ESS R:  0.999 S: 11.988 Class Size: 0.0% (Average: 9.7%)
[Sat, 22 Feb 2025 04:53:12 GMT] [CPU RAM used: 4150 MB]    -- Class  3 -- lr: 0.10 eps: 331867415258755320856599144169472.00 step ratio : 0.0000 ESS R:  1.000 S: 11.990 Class Size: 0.0% (Average: 10.3%)
[Sat, 22 Feb 2025 04:53:12 GMT] [CPU RAM used: 4150 MB]    -- Class  4 -- lr: 0.10 eps: 331867415258755320856599144169472.00 step ratio : 0.0000 ESS R:  1.000 S: 11.990 Class Size: 0.0% (Average: 10.1%)
[Sat, 22 Feb 2025 04:53:12 GMT] [CPU RAM used: 4150 MB]    -- Class  5 -- lr: 0.10 eps: 331867415258755320856599144169472.00 step ratio : 0.0000 ESS R:  1.007 S: 12.000 Class Size: 0.0% (Average: 9.8%)
[Sat, 22 Feb 2025 04:53:12 GMT] [CPU RAM used: 4150 MB]    -- Class  6 -- lr: 0.10 eps: 331867415258755320856599144169472.00 step ratio : 0.0000 ESS R:  1.000 S: 11.994 Class Size: 0.0% (Average: 10.1%)
[Sat, 22 Feb 2025 04:53:12 GMT] [CPU RAM used: 4150 MB]    -- Class  7 -- lr: 0.10 eps: 331867415258755320856599144169472.00 step ratio : 0.0000 ESS R:  1.000 S: 11.994 Class Size: 0.0% (Average: 9.7%)
[Sat, 22 Feb 2025 04:53:12 GMT] [CPU RAM used: 4150 MB]    -- Class  8 -- lr: 0.10 eps: 331867415258755320856599144169472.00 step ratio : 0.0000 ESS R:  0.999 S: 11.988 Class Size: 0.0% (Average: 10.1%)
[Sat, 22 Feb 2025 04:53:12 GMT] [CPU RAM used: 4150 MB]    -- Class  9 -- lr: 0.10 eps: 331867415258755320856599144169472.00 step ratio :   nan ESS R:  1.001 S: 12.000 Class Size: 91.2% (Average: 10.2%)
[Sat, 22 Feb 2025 04:53:18 GMT] [CPU RAM used: 4141 MB]   Done iteration 02683 of 03069 in 15.226s. Total time 38076.3s. Est time remaining 5927.6s.
[Sat, 22 Feb 2025 04:53:18 GMT] [CPU RAM used: 4198 MB] ----------- Iteration  2684 (epoch 1.389).  radwn 48.00  resolution 12.00A  minisize  300  beta 0.00
[Sat, 22 Feb 2025 04:53:26 GMT] [CPU RAM used: 4205 MB]    -- Class  0 -- lr: 0.10 eps: 408168986694449104101698806218752.00 step ratio : 0.0000 ESS R:  1.000 S: 12.000 Class Size: 0.0% (Average: 9.9%)
[Sat, 22 Feb 2025 04:53:27 GMT] [CPU RAM used: 4205 MB]    -- Class  1 -- lr: 0.10 eps: 408168986694449104101698806218752.00 step ratio : 0.0000 ESS R:  0.999 S: 11.994 Class Size: 0.0% (Average: 10.1%)
[Sat, 22 Feb 2025 04:53:27 GMT] [CPU RAM used: 4205 MB]    -- Class  2 -- lr: 0.10 eps: 408168986694449104101698806218752.00 step ratio : 0.0000 ESS R:  1.002 S: 11.987 Class Size: 0.0% (Average: 9.7%)
[Sat, 22 Feb 2025 04:53:27 GMT] [CPU RAM used: 4205 MB]    -- Class  3 -- lr: 0.10 eps: 408168986694449104101698806218752.00 step ratio : 0.0000 ESS R:  1.002 S: 11.998 Class Size: 0.0% (Average: 10.3%)
[Sat, 22 Feb 2025 04:53:27 GMT] [CPU RAM used: 4205 MB]    -- Class  4 -- lr: 0.10 eps: 408168986694449104101698806218752.00 step ratio : 0.0000 ESS R:  1.000 S: 11.990 Class Size: 0.0% (Average: 10.0%)
[Sat, 22 Feb 2025 04:53:27 GMT] [CPU RAM used: 4205 MB]    -- Class  5 -- lr: 0.10 eps: 408168986694449104101698806218752.00 step ratio :   nan ESS R:  1.010 S: 12.000 Class Size: 100.2% (Average: 9.9%)
[Sat, 22 Feb 2025 04:53:27 GMT] [CPU RAM used: 4205 MB]    -- Class  6 -- lr: 0.10 eps: 408168986694449104101698806218752.00 step ratio : 0.0000 ESS R:  1.002 S: 12.000 Class Size: 0.0% (Average: 10.1%)
[Sat, 22 Feb 2025 04:53:27 GMT] [CPU RAM used: 4205 MB]    -- Class  7 -- lr: 0.10 eps: 408168986694449104101698806218752.00 step ratio : 0.0000 ESS R:  1.007 S: 11.999 Class Size: 0.0% (Average: 9.7%)
[Sat, 22 Feb 2025 04:53:27 GMT] [CPU RAM used: 4205 MB]    -- Class  8 -- lr: 0.10 eps: 408168986694449104101698806218752.00 step ratio : 0.0000 ESS R:  1.002 S: 11.988 Class Size: 0.0% (Average: 10.0%)
[Sat, 22 Feb 2025 04:53:28 GMT] [CPU RAM used: 4205 MB]    -- Class  9 -- lr: 0.10 eps: 408168986694449104101698806218752.00 step ratio : 0.0000 ESS R:  1.002 S: 11.989 Class Size: 0.0% (Average: 10.2%)
[Sat, 22 Feb 2025 04:53:33 GMT] [CPU RAM used: 4209 MB]   Done iteration 02684 of 03069 in 14.927s. Total time 38091.3s. Est time remaining 5894.2s.
[Sat, 22 Feb 2025 04:53:34 GMT] [CPU RAM used: 3210 MB] Traceback (most recent call last):
  File "cryosparc_master/cryosparc_compute/run.py", line 116, in cryosparc_master.cryosparc_compute.run.main
  File "cryosparc_master/cryosparc_compute/jobs/abinit/run.py", line 301, in cryosparc_master.cryosparc_compute.jobs.abinit.run.run_homo_abinit
  File "/home/cryosparc_user/cryosparc/cryosparc_worker/cryosparc_compute/noise_model.py", line 119, in get_noise_estimate
    assert n.all(n.isfinite(ret))
AssertionError
(base) [cryosparc_user@localhost ~]$
5

@wtempel
Thank you.

The output as followed.

[spuser@spgpu ~]$ cryosparcm cli “get_job(‘$csprojectid’, ‘$csjobid’, ‘job_type’, ‘version’, ‘instance_information’, ‘status’, ‘params_spec’, ‘errors_run’)”
{‘_id’: ‘67bac722adb1414742bea7a6’, ‘errors_run’: [{‘message’: ‘’, ‘warning’: False}], ‘instance_information’: {‘CUDA_version’: ‘11.8’, ‘available_memory’: ‘230.48GB’, ‘cpu_model’: ‘Intel(R) Xeon(R) Platinum 8358P CPU @ 2.60GHz’, ‘driver_version’: ‘12.0’, ‘gpu_info’: [{‘id’: 0, ‘mem’: 25393692672, ‘name’: ‘NVIDIA GeForce RTX 4090’}], ‘ofd_hard_limit’: 262144, ‘ofd_soft_limit’: 1024, ‘physical_cores’: 64, ‘platform_architecture’: ‘x86_64’, ‘platform_node’: ‘spgpu’, ‘platform_release’: ‘4.18.0-477.15.1.el8_8.x86_64’, ‘platform_version’: ‘#1 SMP Wed Jun 28 15:04:18 UTC 2023’, ‘total_memory’: ‘503.04GB’, ‘used_memory’: ‘267.13GB’}, ‘job_type’: ‘homo_abinit’, ‘params_spec’: {‘abinit_K’: {‘value’: 10}}, ‘project_uid’: ‘P1’, ‘status’: ‘failed’, ‘uid’: ‘J871’, ‘version’: ‘v4.4.1’}

[spuser@spgpu ~]$ cryosparcm eventlog $csprojectid $csjobid | tail -n 40
[Sun, 23 Feb 2025 08:11:31 GMT] [CPU RAM used: 3830 MB] – Class 1 – lr: 0.20 eps: 149124468364881850319559899217920.00 step ratio : 0.0000 ESS R: 0.999 S: 11.986 Class Size: 0.0% (Average: 9.8%)
[Sun, 23 Feb 2025 08:11:31 GMT] [CPU RAM used: 3830 MB] – Class 2 – lr: 0.20 eps: 149124468364881850319559899217920.00 step ratio : 0.0000 ESS R: 1.006 S: 12.067 Class Size: 0.0% (Average: 10.3%)
[Sun, 23 Feb 2025 08:11:31 GMT] [CPU RAM used: 3830 MB] – Class 3 – lr: 0.20 eps: 149124468364881850319559899217920.00 step ratio : 0.0000 ESS R: 0.999 S: 11.988 Class Size: 0.0% (Average: 9.9%)
[Sun, 23 Feb 2025 08:11:31 GMT] [CPU RAM used: 3830 MB] – Class 4 – lr: 0.20 eps: 149124468364881850319559899217920.00 step ratio : 0.0000 ESS R: 0.999 S: 11.987 Class Size: 0.0% (Average: 10.0%)
[Sun, 23 Feb 2025 08:11:32 GMT] [CPU RAM used: 3830 MB] – Class 5 – lr: 0.20 eps: 149124468364881850319559899217920.00 step ratio : 0.0000 ESS R: 1.000 S: 11.995 Class Size: 0.0% (Average: 9.7%)
[Sun, 23 Feb 2025 08:11:32 GMT] [CPU RAM used: 3830 MB] – Class 6 – lr: 0.20 eps: 149124468364881850319559899217920.00 step ratio : 0.0000 ESS R: 1.005 S: 11.995 Class Size: 0.3% (Average: 9.7%)
[Sun, 23 Feb 2025 08:11:32 GMT] [CPU RAM used: 3830 MB] – Class 7 – lr: 0.20 eps: 149124468364881850319559899217920.00 step ratio : 0.0000 ESS R: 0.998 S: 11.977 Class Size: 0.0% (Average: 10.3%)
[Sun, 23 Feb 2025 08:11:32 GMT] [CPU RAM used: 3830 MB] – Class 8 – lr: 0.20 eps: 149124468364881850319559899217920.00 step ratio : nan ESS R: 1.005 S: 11.996 Class Size: 85.5% (Average: 10.3%)
[Sun, 23 Feb 2025 08:11:32 GMT] [CPU RAM used: 3830 MB] – Class 9 – lr: 0.20 eps: 149124468364881850319559899217920.00 step ratio : nan ESS R: 1.011 S: 11.999 Class Size: 14.4% (Average: 10.0%)
[Sun, 23 Feb 2025 08:11:34 GMT] [CPU RAM used: 3830 MB] Done iteration 00831 of 03997 in 10.983s. Total time 4312.2s. Est time remaining 37883.2s.
[Sun, 23 Feb 2025 08:11:34 GMT] [CPU RAM used: 3830 MB] ----------- Iteration 832 (epoch 0.229). radwn 41.57 resolution 13.71A minisize 300 beta 0.00
[Sun, 23 Feb 2025 08:11:43 GMT] [CPU RAM used: 3830 MB] – Class 0 – lr: 0.20 eps: 170562109323817105244761387696128.00 step ratio : nan ESS R: 1.000 S: 11.996 Class Size: 9.0% (Average: 10.0%)
[Sun, 23 Feb 2025 08:11:43 GMT] [CPU RAM used: 3830 MB] – Class 1 – lr: 0.20 eps: 170562109323817105244761387696128.00 step ratio : 0.0000 ESS R: 1.000 S: 11.989 Class Size: 0.0% (Average: 9.8%)
[Sun, 23 Feb 2025 08:11:43 GMT] [CPU RAM used: 3830 MB] – Class 2 – lr: 0.20 eps: 170562109323817105244761387696128.00 step ratio : 0.0000 ESS R: 1.000 S: 12.000 Class Size: 0.0% (Average: 10.3%)
[Sun, 23 Feb 2025 08:11:43 GMT] [CPU RAM used: 3830 MB] – Class 3 – lr: 0.20 eps: 170562109323817105244761387696128.00 step ratio : 0.0000 ESS R: 1.001 S: 11.985 Class Size: 0.0% (Average: 9.9%)
[Sun, 23 Feb 2025 08:11:43 GMT] [CPU RAM used: 3830 MB] – Class 4 – lr: 0.20 eps: 170562109323817105244761387696128.00 step ratio : 0.0000 ESS R: 1.000 S: 11.989 Class Size: 0.0% (Average: 10.0%)
[Sun, 23 Feb 2025 08:11:43 GMT] [CPU RAM used: 3830 MB] – Class 5 – lr: 0.20 eps: 170562109323817105244761387696128.00 step ratio : 0.0000 ESS R: 1.000 S: 11.995 Class Size: 0.0% (Average: 9.7%)
[Sun, 23 Feb 2025 08:11:44 GMT] [CPU RAM used: 3830 MB] – Class 6 – lr: 0.20 eps: 170562109323817105244761387696128.00 step ratio : nan ESS R: 1.000 S: 12.003 Class Size: 91.0% (Average: 9.9%)
[Sun, 23 Feb 2025 08:11:44 GMT] [CPU RAM used: 3830 MB] – Class 7 – lr: 0.20 eps: 170562109323817105244761387696128.00 step ratio : 0.0000 ESS R: 0.999 S: 11.988 Class Size: 0.0% (Average: 10.3%)
[Sun, 23 Feb 2025 08:11:44 GMT] [CPU RAM used: 3830 MB] – Class 8 – lr: 0.20 eps: 170562109323817105244761387696128.00 step ratio : 0.0000 ESS R: 0.999 S: 11.986 Class Size: 0.0% (Average: 10.3%)
[Sun, 23 Feb 2025 08:11:44 GMT] [CPU RAM used: 3830 MB] – Class 9 – lr: 0.20 eps: 170562109323817105244761387696128.00 step ratio : 0.0000 ESS R: 1.002 S: 11.996 Class Size: 0.0% (Average: 9.9%)
[Sun, 23 Feb 2025 08:11:46 GMT] [CPU RAM used: 3830 MB] Done iteration 00832 of 03997 in 12.232s. Total time 4324.5s. Est time remaining 37954.3s.
[Sun, 23 Feb 2025 08:11:47 GMT] [CPU RAM used: 3830 MB] ----------- Iteration 833 (epoch 0.230). radwn 41.61 resolution 13.70A minisize 300 beta 0.00
[Sun, 23 Feb 2025 08:11:55 GMT] [CPU RAM used: 3830 MB] – Class 0 – lr: 0.20 eps: 216232759636337965244262719160320.00 step ratio : nan ESS R: 1.002 S: 11.996 Class Size: 100.2% (Average: 10.2%)
[Sun, 23 Feb 2025 08:11:55 GMT] [CPU RAM used: 3830 MB] – Class 1 – lr: 0.20 eps: 216232759636337965244262719160320.00 step ratio : 0.0000 ESS R: 1.000 S: 11.997 Class Size: 0.0% (Average: 9.8%)
[Sun, 23 Feb 2025 08:11:55 GMT] [CPU RAM used: 3830 MB] – Class 2 – lr: 0.20 eps: 216232759636337965244262719160320.00 step ratio : 0.0000 ESS R: 1.000 S: 11.999 Class Size: 0.0% (Average: 10.2%)
[Sun, 23 Feb 2025 08:11:55 GMT] [CPU RAM used: 3830 MB] – Class 3 – lr: 0.20 eps: 216232759636337965244262719160320.00 step ratio : 0.0000 ESS R: 0.999 S: 11.989 Class Size: 0.0% (Average: 9.9%)
[Sun, 23 Feb 2025 08:11:55 GMT] [CPU RAM used: 3830 MB] – Class 4 – lr: 0.20 eps: 216232759636337965244262719160320.00 step ratio : 0.0000 ESS R: 1.002 S: 11.994 Class Size: 0.0% (Average: 10.0%)
[Sun, 23 Feb 2025 08:11:55 GMT] [CPU RAM used: 3830 MB] – Class 5 – lr: 0.20 eps: 216232759636337965244262719160320.00 step ratio : 0.0000 ESS R: 1.003 S: 11.998 Class Size: 0.0% (Average: 9.7%)
[Sun, 23 Feb 2025 08:11:56 GMT] [CPU RAM used: 3830 MB] – Class 6 – lr: 0.20 eps: 216232759636337965244262719160320.00 step ratio : 0.0000 ESS R: 1.000 S: 11.991 Class Size: 0.0% (Average: 9.9%)
[Sun, 23 Feb 2025 08:11:56 GMT] [CPU RAM used: 3830 MB] – Class 7 – lr: 0.20 eps: 216232759636337965244262719160320.00 step ratio : 0.0000 ESS R: 0.999 S: 11.986 Class Size: 0.0% (Average: 10.2%)
[Sun, 23 Feb 2025 08:11:56 GMT] [CPU RAM used: 3830 MB] – Class 8 – lr: 0.20 eps: 216232759636337965244262719160320.00 step ratio : 0.0000 ESS R: 0.999 S: 11.987 Class Size: 0.0% (Average: 10.3%)
[Sun, 23 Feb 2025 08:11:56 GMT] [CPU RAM used: 3830 MB] – Class 9 – lr: 0.20 eps: 216232759636337965244262719160320.00 step ratio : 0.0000 ESS R: 1.005 S: 11.997 Class Size: 0.0% (Average: 9.9%)
[Sun, 23 Feb 2025 08:11:58 GMT] [CPU RAM used: 3830 MB] Done iteration 00833 of 03997 in 11.593s. Total time 4336.1s. Est time remaining 37815.0s.
[Sun, 23 Feb 2025 08:12:00 GMT] [CPU RAM used: 3138 MB] Traceback (most recent call last):
File “cryosparc_master/cryosparc_compute/run.py”, line 95, in cryosparc_master.cryosparc_compute.run.main
File “cryosparc_master/cryosparc_compute/jobs/abinit/run.py”, line 285, in cryosparc_master.cryosparc_compute.jobs.abinit.run.run_homo_abinit
File “/spshared/apps/cryosparc4/cryosparc_worker/cryosparc_compute/noise_model.py”, line 119, in get_noise_estimate
assert n.all(n.isfinite(ret))
AssertionError

6

@T_Bird

thank you for your reply.

I have manully stopped the Ab-initio job as per your advice, and then did a hetero refine with a map that seems good in Ab-initio.

It worked fine, but th results were not good, lots of strange map, probably caused by poor particle quality.

7

Has anyone ever reconstructed 80S ribosome using cryosparc and why does my ribosome have a such severe orientation bias.

I have found a lot of articles about ribosome, but none have ever reported orientation bias, and it seems I am the only one with this problem.

sad

8

Hi, normally we use grids with 2nm carbon for ribosome samples and no preferred orientation seen ever.

9

@xaoshi

Thank you for your reply !

I am using the quantifoil r 1.2/1.3 mesh grid. Maybe it not caused by the type of grid.

I am still don’t understand why my ribosome purified with sucrose cushion also has an orientation bias, there are only hepes, kcl, mgcl2, dtt in the buffer.

Is it because my behaviour when sample preparation for cryo-EM? (vitrobot 100 humidity 4℃)

10

That’s what I thought. Quantifoil R1.2/1.3 with 2nm carbon support is proved to be ideal for ribosome samples. Just try it out.

An example:

image
image1010×206 35.6 KB

11

@xaoshi

This is my select 2D, it seems that too many side view has been picked

J870_selected_14_classes
J870_selected_14_classes1010×206 50.5 KB

and next figure is worked Ab-initio job output.

J873_structure_for_class_000_iteration_3996
J873_structure_for_class_000_iteration_39961057×270 83.4 KB

J873_structure_for_class_001_iteration_3996
J873_structure_for_class_001_iteration_39961057×270 89 KB

J873_structure_for_class_002_iteration_3996
J873_structure_for_class_002_iteration_39961057×273 75.4 KB

J873_structure_for_class_003_iteration_3996
J873_structure_for_class_003_iteration_39961057×270 84.4 KB

12

In your case, ribosomes prefer staying at the air-water interface, which would probably cause preferential orientation. 2 nm Carbon support could improve sample adsorption and prevent preferential orientation.

13

Hi

Thank you for you advise!

I used +2nm carbon film to prepare ribosome samples. My glow discharge conditions were 60 seconds glow time and 10 seconds hold time. It seems the carbon film is prone to rupture. May I ask what treatment conditions you typically use for this type of carbon film?

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@hai for commercial thin carbon grids I would try a gentle treatment starting around 10 mA for 10 seconds.