Bin micrographs

Cryo-EM Data Processing
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A post was merged into an existing topic: No particles from filament tracer

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Hi Oli and wtempel,

The above issue also taken care of at present (BUT I will revisit it later to provide you more details). I have another problem.

  1. I recently completely a helical reconstruction with resolution up to 3.6Å. Now I want to generate the whole 3.6Å map to a much larger length (it is a phage tail density map). I will need the asymmetric map (at 3.6Å) so that I can symmetrize it to generate a 1200Å long map (the map is ~256Å long). How to obtain the asymmetric map ?

Is there any alternate way to do it ?

  1. I want to sort out heterogeneity based on the Pitch of the helix. I can generate maps for classifications from the asymmetric map, requested above. Following that How to I carry out the classification using the maps in cryosparc ?

Thanks

Andy

How do I do that?

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Hi @Andy,
Thank you for posting these interesting questions.

To facilitate use and management of this forum, please post questions 1. and 2. separately as two new topics in the Helical Processing category.

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Hi Andy,

Can I know how to bin micrographs in the CS? What’s the job name?

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Hi

I binned the images in BSOFT (or you can do it in any package like EMAN2) and then imported them in Cryosparc.

However if memory serves well, there should be an option in cryosparc, I can possibly reply you back tomorrow or day after tomorrow. Some one from cryosparc team can definitely reply you, sooner but you can try the ‘Fourier crop to box size’.

Best

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Depending on your stage of the workflow, “binning” can be accomplished in various job types.
For example, see this discussion about Patch Motion Correction.
Additionally, there are the Extract From Micrographs and Downsample Particles job types that can achieve a binning effect.

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Hi Wtempel,

I’m running the Topaz Extract job, but it errored “out of memory”. I thought if I can bin the micrograph in a separate job, then do the Topaz extract, this error may cleared.

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Hi @CleoShen - the defaults for topaz extract are quite memory hungry - you might try reducing the number of threads and processes, if you haven’t already.

Cheers
Oli

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Hi Oli,

Thank you for your reply.
I binned the micrographs by Patch Motion Cor Job, then the Topaz Extract works, but I would like to downsample more, such as bin8, bin16, but the CS Patch Motion Cor F-crop option only provides as many as bin4, if you know how can I downsample more in CS?

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This shouldn’t be necessary to run Topaz, as Topaz downsamples on the fly. Have you tried adjusting the downsampling factor in Topaz? What settings are you using?

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Do you require down-sampling beyond factor 4 before picking, or would a downstream Downsample Particles job work for you?

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@CleoShen
you may want to use some other package to downsample of desired value. What stage of processing you are in ?

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@olibclarke @wtempel
I am taking this thread to ask about the helical processing questions that I have posted 2 months ago ? I think a separate thread is made but have not received any response. Can you help.

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I have no idea, I have not worked much with helical samples, sorry…

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Yes, I’d like to down-sample 8 before picking. I thought I could do two rounds of bin when running two times of patch motion cor, but it doesn’t work.

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I’m running the Topaz extract job on bin4 micrograph now.

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Hi @CleoShen you do not need to downsample your micrographs for Topaz. This happens automatically during preprocessing in Topaz, and is controlled by the downsampling factor that you set in Topaz train. What settings are you using for Topaz train/extract?

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Also can you post the error you are seeing in Topaz extract? If you are trying to run Topaz extract on a lot of micrographs (>5k), then you will need to split into smaller groups for Topaz extract

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Hi Oli,

I set downsample4 at the beginning, but the jobs are killed multiple times (I assume it’s because of the big memory request); then I tested downsampling by patch motion cor first, then the Topaz extra works.

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Can you post the error, and the settings you are using