[3dem] Reconstructing a K3 tomogram

Mon Feb 28 07:42:28 PST 2022

Hi Joanna,
while I'm sure there are IMOD solutions to this problem, you might consider trying:

https://urldefense.proofpoint.com/v2/url?u=https-3A__eman2.org_e2TomoSmall&d=DwIGaQ&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=6N7sfHZaBpcerWmtN1_-l1d04aKUt54QeF2j5nE49l3TFxgEzKxiLQEVD_BL4zB2&s=MwUubCDvPDj6bqvz73Cdcv689W8nOTEYvmhTiLGYM48&e= 

which is fully automated and will produce downsampled tomograms by default, but return to the fully sampled data if you extract local regions (for example for subtomogram averaging).

--------------------------------------------------------------------------------------
Steven Ludtke, Ph.D. <sludtke at bcm.edu<mailto:sludtke at bcm.edu>>                      Baylor College of Medicine
Charles C. Bell Jr., Professor of Structural Biology
Dept. of Biochemistry and Molecular Biology                      (https://urldefense.proofpoint.com/v2/url?u=http-3A__www.bcm.edu_biochem&d=DwIGaQ&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=6N7sfHZaBpcerWmtN1_-l1d04aKUt54QeF2j5nE49l3TFxgEzKxiLQEVD_BL4zB2&s=7oxCscH0XQv55BzHQ56DytgpGhs5nGDErj49B3U4T50&e= <https://urldefense.proofpoint.com/v2/url?u=http-3A__www.bcm.edu_biochem&d=DwIGaQ&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=6N7sfHZaBpcerWmtN1_-l1d04aKUt54QeF2j5nE49l3TFxgEzKxiLQEVD_BL4zB2&s=7oxCscH0XQv55BzHQ56DytgpGhs5nGDErj49B3U4T50&e= >)
Academic Director, CryoEM Core                                        (cryoem.bcm.edu<https://urldefense.proofpoint.com/v2/url?u=http-3A__cryoem.bcm.edu&d=DwIGaQ&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=6N7sfHZaBpcerWmtN1_-l1d04aKUt54QeF2j5nE49l3TFxgEzKxiLQEVD_BL4zB2&s=c3N_c2MswvfkmffWYmrECnGnp16qPZ76-SmY2EMzlXw&e= >)
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On Feb 28, 2022, at 9:33 AM, Joanna Brown <joanna.brown at astx.com<mailto:joanna.brown at astx.com>> wrote:

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________________________________
Hello everyone,

I’m looking for some help reconstructing a tomogram taken on a Krios with a K3 detector.

I’m doing the reconstruction with imod/etomo v4.9. I’ve taken a relatively low mag tomogram of a 0.6/1 quantifoil hole with particles in it so I can see the particle distribution and ice thickness. I can see the whole foilhole in the field of view.

I am using patch tracking and everything looks fine with the aligned stacks until I come to the point in the process where a tomogram needs to be computed, including making the sample tomogram to generate the boundary model. (At least I think it does, but I have to admit, I’m pretty rusty with imod/etomo!)

I notice that the X and the Z axis seem to become switched and I end up with a tomogram of over 1500 Z slices that corresponds to the pixel dimensions of the K3 detector. The reconstruction also becomes very strange, the protein particles (these look reasonable though strangely arranged relative to each other) visible in a separate plane from the foil hole, which ends up looking like an eye shape instead of a round hole. I tried to compute the tomogram using WBP.

Thoughts I  have had to try to fix problem are:

  1.  Try making an initial X,Y boundary model to only include the foil hole for patch tracking and exclude all the carbon and very small particles.
  2.  Try a low pass filter?
  3.  No idea what to do about the swapping of X and Z dimensions – help!

Any suggestions or pointing out my obvious glaring error/stupidity totally welcome!

Thanks so much!

Joanna Brown

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