Pixel size after tomogram reconstruction becomes 1?

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yang...@yale.edu

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Apr 20, 2021, 5:19:49 PM4/20/21
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Dear Steve and EMAN2 team,
I have a naive question. I noticed that the pixel size after tomogram reconstruction becomes 1 A (read from the header), while my original tiltseries was 2.1 A. I don't remember I specified the pixel size at any step and I used bin 4 (1k) for tomogram generation, so I should expect a pixel size around 8.4 A, right? Because I noticed that all my downstream particle pixel size also was 1 A. I am curious if this is something I need to pay attention to? If so, how should I fix it? 
Best,
Yangqi

digvij...@gmail.com

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Apr 21, 2021, 1:35:50 AM4/21/21
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Hi Yangqi,

I am not from the EMAN2 team, so you may probably get more accurate answer(s) from the EMAN2 team. 
Here's what I think may be happening. 
  • Check the pixel size of your tiltseries. When importing tilt-series into the EMAN2 (under the Raw Data tab), there is an option to specify the correct pixel size (under the apix field). Usually, EMAN2 is able to automatically read the pixel size from the header of the raw tilt-series stack when importing the tilt-series. It may be possible that EMAN2 could not read the pixel size from your tilt-series while importing it or you specifed apix of 1 while importing. 
  • Re-import the tilt-series and specify the correct pix size (unbinned) and when you re-reconstruct, the tomograms should have the right pixel size. 

    Cheers,
    Digvijay

Yangqi Gu

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Apr 21, 2021, 12:47:56 PM4/21/21
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Hi Digvijay,
Thank you for your reply. I did check the tiltseries after import, they look okay with the right pixel size. So I went on to extract particles and did some 3D refinement. I used a bin2 for my particles since they are pretty big (350 pixel for unbinned). It looks all okay with the right shape. But when I re-extracted the particles with unbinned size and did the refinement, it all looked messed-up. So I was wondering if the pixel size is somehow messing up the refinement.
Best,
Yangqi


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Yangqi Gu
Graduate Student
Malvankar Lab
Yale University, West Campus

Muyuan Chen

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Apr 21, 2021, 1:07:00 PM4/21/21
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I don't quite understand why this happened either. The particles should really follow the pixel size of tilt series... When you look at the particles after re-extraction, do they have the right apix? Or is that only the maps in the refinement have the apix=1? Specifically, at which step does the apix change?
Sometimes strange behavior can happen when particles of pixel sizes are combined to the same refinement, but even that shouldn't set the pixel size back to one... 

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Yangqi Gu

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Apr 21, 2021, 1:39:55 PM4/21/21
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Hi Muyuan,
The pixel changed after I obtained the tomogram (bin4). It became 1 and all the following steps became 1. Strangely, after I did the refinement with bin2 particles, I performed the e2proc3d.py --scale=2 to unbin the map, the pixel size went back to 2.1 for the map (seems doing the right job). But when I re-extracted the particles unbinned, the unbinned particles became 1 again. But I can run the program fine, at least it did not complain about pixel size mismatch with the reference (unbinned map). So I was quite confused.
Best,
Yangqi

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Muyuan Chen

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Apr 21, 2021, 1:49:44 PM4/21/21
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How did you read the apix value? Can you run "e2iminfo.py -H xxx.hdf" for the tilt series, tomogram, 3d particle and the reference and check the apix_x line? 

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Yangqi Gu

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Apr 21, 2021, 2:10:14 PM4/21/21
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Hi Muyuan,
When I used e2iminfo.py, it did not output the information about pixel size except stack size and how many images. So I used header from IMOD to read the header of .hdf file. 
Best,
Yangqi

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Ludtke, Steven J.

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Apr 21, 2021, 2:16:37 PM4/21/21
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HDF support is quite new to IMOD and I'm not sure if there is a perfect metadata match. Adding the -H option to e2iminfo.py will list the complete header.

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Steven Ludtke, Ph.D. <slu...@bcm.edu>                      Baylor College of Medicine 
Charles C. Bell Jr., Professor of Structural Biology
Dept. of Biochemistry and Molecular Biology                      (www.bcm.edu/biochem)
Academic Director, CryoEM Core                                        (cryoem.bcm.edu)
Co-Director CIBR Center                                    (www.bcm.edu/research/cibr)




Yangqi Gu

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Apr 21, 2021, 4:01:23 PM4/21/21
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Hi Steve,
It seems indeed the header mismatch problem. Now the pixel size is correct output by e2iminfo.py. Thank you!
Best,
Yangqi
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Yangqi Gu
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Malvankar Lab
Yale University, West Campus

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Yangqi Gu
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Malvankar Lab
Yale University, West Campus

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Yangqi Gu
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