issue with 9 optics groups during polishing

[ashkumatov]

Hi
In my dataset i have 9 optics groups. In half of them px size is 0.784 and in the other half 0.764. All datasets have been extracted with box size of 400px and decimated to 200px.
When i try polishing, i get the error below. I have done smth similar with 5 optics groups previously (also px size was varying), so could it be a bug or certain limitation on number of optics groups?
if i use the option '--par_group 1' to estimated motion for the first group, it takes actually micrographs from various OGs despite my expectations.

Could you comment on this? possible bug? let me know if any further details are needed.

Thank you in advance
Alex

WARNING: You did not specify --angpix_ref. The pixel size in the image header of Refine3D/job073/run_half1_class001_unfil.mrc, 1.528 A/px, is used.
in: /home/software/relion/git-relion-3.1.0/src/jaz/motion/motion_param_estimator.cpp, line 101
ERROR: 
MotionParamEstimator::init: unable to estimate motion parameters for all 
optics groups simultaneously due to varying pixel and box sizes.
Please estimate them separately for each optics group (--par_group).
=== Backtrace  ===
/home/software/relion/git-relion-3.1.0/build-relion3.1.0-20200707_cu92/bin/relion_motion_refine(_ZN11RelionErrorC2ERKNSt7__cxx1112basic_stringIcSt11char_traitsIcESaIcEEES7_l+0x77) [0x55a9d6c42117]
/home/software/relion/git-relion-3.1.0/build-relion3.1.0-20200707_cu92/bin/relion_motion_refine(_ZN20MotionParamEstimator4initEiibNSt7__cxx1112basic_stringIcSt11char_traitsIcESaIcEEEiRKSt6vectorI13MetaDataTableSaIS7_EEP15MotionEstimatorP12ReferenceMapP16ObservationModel+0xddc) [0x55a9d6d8902c]
/home/software/relion/git-relion-3.1.0/build-relion3.1.0-20200707_cu92/bin/relion_motion_refine(_ZN13MotionRefiner4initEv+0x104d) [0x55a9d6cd31cd]
/home/software/relion/git-relion-3.1.0/build-relion3.1.0-20200707_cu92/bin/relion_motion_refine(main+0x47) [0x55a9d6c3cb97]
/lib/x86_64-linux-gnu/libc.so.6(__libc_start_main+0xe7) [0x7f3971f84b97]
/home/software/relion/git-relion-3.1.0/build-relion3.1.0-20200707_cu92/bin/relion_motion_refine(_start+0x2a) [0x55a9d6c3d92a]
==================
ERROR: 
MotionParamEstimator::init: unable to estimate motion parameters for all 
optics groups simultaneously due to varying pixel and box sizes.
Please estimate them separately for each optics group (--par_group).

[biochem-fan]

I guess you have merged two datasets. In this case, you should polish them separately.

See https://www3.mrc-lmb.cam.ac.uk/relion/index.php/Pixel_size_issues#How_can_I_merge_datasets_with_different_pixel_sizes.3F.

[ashkumatov]

that depends how one defines a 'dataset': i have one sample collected from two grids over a period of ~2 weeks on the the same microscope/detector intermittent by microscope calibration, alignment, etc, which resulted in 9 optics groups.
Do i understand it correctly that you suggest to split the Refine3D/run_data.star into 2 parts with similar px size and run Polishing on the them?

[biochem-fan]

i have one sample collected from two grids over a period of ~2 weeks on the the same microscope/detector intermittent by microscope calibration, alignment

In this case, we consider them different datasets, because the physical pixel size is different.
Moreover, the dose rate might have drifted by re-alignment (note that the dose rate displayed on EPU or DM is not very accurate). So you have to polish them separately.

split the Refine3D/run_data.star into 2 parts

You don't have to split run_data.star. As written in the page above, keep MotionCorr STAR files separate and run two Polish jobs, followed by JoinStar.

[ashkumatov]

In this case, we consider them different datasets, because the physical pixel size is different.
Moreover, the dose rate might have drifted by re-alignment (note that the dose rate displayed on EPU or DM is not very accurate). So you have to polish them separately.

in this case you consider every OG as a separate dataset? or you mean only 2 datasets (based on px size in the OG)?

You don't have to split run_data.star. As written in the page above, keep MotionCorr STAR files separate and run two Polish jobs, followed by JoinStar.

sorry, just to be clear: i have OG1-9 from which OG1 to OG5 have px size of X and OG6 to OG9 have px size Y. Currently, i have Refine3D/ job ( after CTFrefinement ) with 9 OGs that i would like to polish. If i understand correctly, I should provide the Refine3D/run_data.star and MC2 file corresponding to OG1-5 (Polish job #1) and to OG6-9 (Polish job #2).

Thank you for clarifications!

[biochem-fan]

in this case you consider every OG as a separate dataset? or you mean only 2 datasets (based on px size in the OG)?

At least you have to consider optics groups with different pixel sizes (i.e. OG1-5 vs OG6-9) as different datasets. Whether you should polish even more separately for every dataset (OG1, OG2, ...) depends on what you changed between them, but in general, it is not necessary.

i have OG1-9 from which OG1 to OG5 have px size of X and OG6 to OG9 have px size Y. Currently, i have Refine3D/ job ( after CTFrefinement ) with 9 OGs that i would like to polish. If i understand correctly, I should provide the Refine3D/run_data.star and MC2 file corresponding to OG1-5 (Polish job #1) and to OG6-9 (Polish job #2).

Yes.

[ashkumatov]

You don't have to split run_data.star. As written in the page above, keep MotionCorr STAR files separate and run two Polish jobs, followed by JoinStar.

If i have a MC2 file for one datasets (OGs with same px size) and provide run_data.star (from 2 datasets as described above), i still get the error. No error, when run_data.star is split manually

[biochem-fan]

This is unexpected and I don't know why it happens, but you can workaround as you said.