Import tomograms from IMOD

[Anton Avramov]

Steve, 

I do apologize for bombarding you with the questions... 

Here is my next problem, I hope you will be able to help me. I had to use IMOD to reconstruct the tomograms for some of my tilt series, because EMAN2 was not able to do a good job, I have highly repetitive paracrystalline structures that occupy the entire cell... I feel that EMAN2 couldn't do nice patch tracking, while in IMOD I was able to limit how far each patch can jump on every tilt. However, I do want to use EMAN2 for particle picking, model generation and 3D refinement, because it is so much easier than in IMOD. 

I'm trying to import the tomogram from IMOD into EMAN2 and I'm facing a problem, that I don't even know how to describe.

First issue that I noticed that tilt.json doesn't contain any info about tilt stack file, or tilt_params. 

Second issues, which is very confusing - when I open e2tomo_eval I can see a tomogram but the preview window is showing me preview from Y axis slice and not Z image. If I go into a "Boxer" particle picking also happen in Y slice and not from Z. If I scroll the slider on the right - the image with Y slice will change but Z will stay the same. The first screenshot is the e2tomo_eval preview window. Second from top to bottom: Particle picked up using Z window, same particle on Y window and actual particle picked up. 

I've read your comments where you don't recommend importing tomograms from IMOD, but I hope there is a solution for my problem, because the tomogram from IMOD is perfect for my analysis.

Thank you so much for your help! 

Anton 

[Ludtke, Steven J.]

On Feb 21, 2024, at 4:49 PM, Anton Avramov <anav...@colorado.edu> wrote:

Steve, 

I do apologize for bombarding you with the questions... 

It's fine, that's why we have a mailing list. 

Here is my next problem, I hope you will be able to help me. I had to use IMOD to reconstruct the tomograms for some of my tilt series, because EMAN2 was not able to do a good job, I have highly repetitive paracrystalline structures that occupy the entire cell... I feel that EMAN2 couldn't do nice patch tracking, while in IMOD I was able to limit how far each patch can jump on every tilt. However, I do want to use EMAN2 for particle picking, model generation and 3D refinement, because it is so much easier than in IMOD. 

Unfortunately, there is a limited amount you will be able to to in EMAN2 with an IMOD generated tomogram. You can do the first stage of subtomogram averaging from the "old" pipeline, which is based on 3-D particles without CTF correction (limited resolution). The second stage of the old pipeline and the entire new pipeline don't actually use the 3-D particles extracted from the tomogram at all. Even the '3-D alignment' stage of the new spt refinement is working with 3-D particles reconstructed individually from the tilt series, rather than particles extracted from the tomogram. It is not straigtforward to get the information from the imod alignment into EMAN2.

I'm trying to import the tomogram from IMOD into EMAN2 and I'm facing a problem, that I don't even know how to describe.

First issue that I noticed that tilt.json doesn't contain any info about tilt stack file, or tilt_params. 

Second issues, which is very confusing - when I open e2tomo_eval I can see a tomogram but the preview window is showing me preview from Y axis slice and not Z image. If I go into a "Boxer" particle picking also happen in Y slice and not from Z. If I scroll the slider on the right - the image with Y slice will change but Z will stay the same. The first screenshot is the e2tomo_eval preview window. Second from top to bottom: Particle picked up using Z window, same particle on Y window and actual particle picked up. 

<Screenshot 2024-02-21 at 3.37.40 PM.png>

<Screenshot 2024-02-21 at 3.43.47 PM.png>

I've read your comments where you don't recommend importing tomograms from IMOD, but I hope there is a solution for my problem, because the tomogram from IMOD is perfect for my analysis.

This problem is easily fixable. IMOD generates tomograms along a different axis than most other software. EMAN2 puts the short axis along Z. Both IMOD and EMAN2 have tools you can use to rotate the reconstruction to put the short axis on Z. This would allow you to box out particles and do the first stage refinement mentioned above, but subtilt refinement would be impossible, as would the much improved new refinement pipeline, limiting your resolution to 20-40 A, depending on defocus.

From our perspective, the correct solution to this problem would be to make EMAN2 work properly with your tilt series. I guess your issue is that EMAN2 is shifting the alignment by one or more unit-cells between shifts. If you are willing to share a couple of representative tilt series (privately) we can take a look and see if there are any existing options which might solve your problem. You could try:

- maybe in this situation, disable patch tracking?

- --maxshift MAXSHIFT   Maximum shift between tilts(/image size). default is 0.2.  The units are the size of the image, ie 0.2 is 20% of the image size.

If those don't help, there may be some other possibilities. 

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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 
Deputy Director, Advanced Technology Cores                  and Molecular Pharmacology
Academic Director, CryoEM Core
Co-Director CIBR Center