[3dem] 3dem Digest (combining tilt-series with different mag)

Wed Sep 8 12:45:54 PDT 2021

Hi Mat,

Here are some options, mileage may vary:

- If you want to use Fourier Cropping, you could use the CLI program
"resample" that comes with cisTEM.
- At your resolution, you could probably just use real-space resampling via
IMOD's "newstack -shrink [shrinkFactor]", double check the man page, but I
think you would want a shrinkFactor > 1 applied to your stack with the
higher pixel sampling rate (lower pixel size.) e.g. newstack -shrink 1.035
stack_in.mrc stack_out.mrc. The header should confirm the correct pixel
size (header stack_out.mrc)

   * * * Both of these would change the imod alignments, which are in
pixels. You might just scale the 5/6 column which are x/y shifts from your
imod.xf file, or alternatively, just update your tilt-series automatically
by using "emClarity autoAlign" which will do an automatic tilt-series
alignment for you. If you have more questions about that option, please
post them on the emClarity mailing list.

- Finally, if you are feeling spunky, I think you could get away with
scaling the first 4 columns in your imod.xf files, which are a
transformation matrix that includes magnification. emClarity uses this
matrix in Fourier Space when resampling the tilt-series. Using awk or
something, you would just scale those 4 values by your wanted mag change.
Off the top of my head, I can't remember which you would want, so as a
control, you could take a stack, scale by either 1/2 or 2, and then run
"emClarity ctf estimate" which outputs the resampled stack in your
"aliStacks" directory. It should be clear which direction you need to go
then. Same advice for further questions on this option, as it is emC
specific, please post over that way.

HTH

Ben

------------------------
Benjamin Himes, PhD

cryoEM methods development
HHMI, RNA Therapeutics Institute, University of Massachusetts Medical School

cryo-STA development @ emClarity <https://urldefense.proofpoint.com/v2/url?u=https-3A__github.com_bHimes_emClarity&d=DwIBaQ&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=M_dZB06pzOavKuuu783BOvjiGtUd5r50ihEbVPhfzzs&s=2khunCSL7vOwJCK-LNA1OaAvRBPVrwGQQwF16ZK_tP8&e= >
Visual Proteomics @ cisTEM <https://urldefense.proofpoint.com/v2/url?u=https-3A__github.com_timothygrant80_cisTEM&d=DwIBaQ&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=M_dZB06pzOavKuuu783BOvjiGtUd5r50ihEbVPhfzzs&s=SID-lccuQDLpb6fqKdcvafXXzAdEn5hAnWY5zeOJdU0&e= >
-------------------------

On Wed, Sep 8, 2021 at 3:00 PM <3dem-request at ncmir.ucsd.edu> wrote:

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>    1. Fourier cropping to merge tomography datasets (McLaren, Mathew)
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> Message: 1
> Date: Wed, 8 Sep 2021 11:11:16 +0000
> From: "McLaren, Mathew" <M.McLaren at exeter.ac.uk>
> To: "3dem at ncmir.ucsd.edu" <3dem at ncmir.ucsd.edu>
> Subject: [3dem] Fourier cropping to merge tomography datasets
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> LNXP265MB084195CDCA40B6D0A9B97751C4D49 at LNXP265MB0841.GBRP265.PROD.OUTLOOK.COM
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>
> Dear all,
>
> I am working with two tomogram datasets collected using two separate Krios
> microscopes and I've been picking particles to perform STA. I have somewhat
> successfully processed these using Relion to a resolution of 20 A but I've
> been experimenting with emClarity in an attempt to improve it further.
> There is a small pixel size difference (4.377 A and 4.53 A), which I've
> previously accounted for by resizing the processed tomogram with the
> smaller pixel size through IMOD to match the larger one. I was looking for
> a different approach to this as I realise that this is probably a terrible
> way to combine two datasets!
>
> For emClarity I need to work with the tilt series, so I was thinking that
> I should do a (non-integer) fourier crop the tilts from one dataset so they
> match with the other, and I was wondering if anyone has any experience of
> the best way to do this? Also, would this invalidate the alignments
> performed in IMOD and require me to reprocess the tilt series?
>
> Cheers,
>
> Mat
>
>
> ----------
>
>
> Dr Mathew McLaren
>
> TEM Experimental Officer
>
> Living Systems Institute
>
> University of Exeter
>
> T: +44(0)1392 727468
>
> E: m.mclaren at exeter.ac.uk
>
>
> My pronouns are he / him / his.
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