Aretomo Alignments and Warp Reconstructions

[Kyle Messina]

Hello,

Currently I've been trying to use Aretomo and Aretomo2 for alignments and the results have been pretty bad with almost no contrast and poor looking features.  As a control, I used Aretomo and Aretomo2 on the raw tilt stacks from the Krios and the reconstructions look great.  So I assume the issue is either the arguments I'm using with Aretomo for the alignments or I'm somehow messing up the import.  I have had no issues with IMOD, so any Aretomo advice is appreciated.

I haven't tried to use Aretomo to reconstruct the tilt stacks made by WarpTools yet so I'll try that after this post just to see as another control.

Thank you,

Kyle Messina

[Warp Bot]

Hi Kyle,

This is a common issue when using AreTomo externally and importing alignments into WarpTools. A few things to check:

1. Are you running AreTomo on the tilt stacks generated by WarpTools (via ts_stack), or on the raw stacks from the microscope? If you are running AreTomo on the WarpTools-generated stacks, make sure --alignment_angpix in ts_import_alignments matches the pixel size of those stacks exactly.

2. When running AreTomo, make sure you use -TiltCor -1 (or 0). If AreTomo corrects tilt angles and you do not update your mdoc files to match, Warp will apply the wrong angles and the reconstruction will look bad.

3. Use -OutImod 2 so AreTomo outputs the .xf file in the format Warp expects.

4. AreTomo uses SART reconstruction by default, which looks much more contrasty than Warp's CTF-corrected weighted back projection. Some of the contrast difference you are seeing may be expected. Try adding --deconv when running ts_reconstruct to improve contrast in the WarpTools output.

5. Consider using the built-in WarpTools wrapper (ts_aretomo3) instead of running AreTomo externally. The wrapper handles pixel size conversion, alignment import, and tilt angle management automatically, which eliminates most of these pitfalls.

To help narrow things down further, could you share:
- The exact WarpTools and AreTomo commands you are running?
- What pixel size your tilt stacks are at, and what value you are passing to --alignment_angpix?
- Which AreTomo version you are using?

-- Warp Bot
THIS IS AN AUTOMATED MESSAGE GENERATED BY AN LLM. IT MAY OR MAY NOT SOLVE YOUR PROBLEM. IF YOU'D LIKE TO SPEAK TO A HUMAN, SAY SO IN YOUR MESSAGE.

--
You received this message because you are subscribed to the Google Groups "Warp" group.
To unsubscribe from this group and stop receiving emails from it, send an email to warp-em+u...@googlegroups.com.
To view this discussion visit https://groups.google.com/d/msgid/warp-em/40e07ed0-2502-4bac-859d-41cca89f0075n%40googlegroups.com.

[Kyle Messina]

I had someone reach out to me, before the bot, and I was able to proceed in the reconstruction with their advice. Turns out updating to the newest WarpTools build (38) and using Aretomo3 fixed all my issues.  I had no issues using the Wrapper or using the ts_stack option and using Aretomo3 outside WarpTools.  The alignments looked a bit better without using the Wrapper but they were essentially the same. 

-Kyle