Difference in alignment quality between StandAlone Aretomo3 and the Aretomo3 wrapper inside WarpTools

[Maurice Frijns]

Hi everyone, 

I am curious if others have seen similar results :)

I collected some tiltseries on P97 (hexameric ~600 kDa protein) and used warptools v36 with the aretomo3 wrapper (2.2.2) for alignment using the option to determine the tilt axis from 4 tiltseries with 4 itterations. 

Next to that I did the exact same processing steps in warptools but did the alignment using standalone aretomo3 2.2.2 and imported those into warptools and did the same other processing steps resulting in the screenshot below. 

On the left (with banana gold) is from the Aretomo3 wrapper inside warptools and right (good looking gold) from standalone aretomo3.

I am currently thinking that something might be off when determining the tilt axis angle as these are slightly different between the wrapper and standalone aretomo3 however I don't interfere there in both as I let the software determine it by themselfs. 

Curious to hear if others have had similar results or a possible fix?

Cheers,

Maurice 

[Marten Chaillet]

Hi Maurice,

What are the values for the tilt axis angle in the .xml for both those tomograms? 

Cheers,
Marten

Op vrijdag 16 januari 2026 om 15:17:27 UTC+1 schreef m.a.fri...@gmail.com:

[Maurice Frijns]

Hi Marten,

When I import AreTomo3 alignments from Standalone Aretomo 3 2.2.2 the value is -89.828 and for the wrapper of aretomo3 inside warptools it is -88.682.

I run a quick test and think that between Warptools 2.0.0 and version 2.0.0dev36 there might be some kind of bug.  

I made a fresh folder with the mdoc and frames for one tiltseries and as can be seen in the command below. first I run the command with the older warptools version and removed the entire tiltstack folder followed by running the command in the newer warptools version. Basically it doesn't refine the tilt axis angle anymore as can be seen in the command below. Hopefully I explained understandably :)

Cheers,

Maurice 

 

(warptools) /data2A/mfrijns/CD25VCP/TestWarpTools$ srun -n 1 --cpus-per-task 12 --partition=gpuq --gres=gpu:4 WarpTools  ts_aretomo --settings warp_tiltseries.settings --angpix 10 --axis_iter 1 --axis_batch 1 --exe /software/apps/aretomo/1.3.4/aretomo --alignz 1000
Running command ts_aretomo with:
angpix = 10
mask = False
alignz = 1000
axis_iter = 1
axis_batch = 1
min_fov = 0
axis = null
patches = null
delete_intermediate = False
exe = /software/apps/aretomo/1.3.4/aretomo
device_list = {  }
perdevice = 1
workers = {  }
settings = warp_tiltseries.settings
input_data = {  }
input_data_recursive = False
input_processing = null
output_processing = null
input_norawdata = False

No alternative input specified, will use input parameters from warp_tiltseries.settings
File search will be relative to /data2A/mfrijns/CD25VCP/TestWarpTools/tomostar
1 files found
Parsing previous results for each item, if available...
1/1, previous metadata found for 0
Connecting to workers...
Connected to 1 workers
Current tilt axis angle: -88.700 °
Running iteration 1 of tilt axis refinement:
Using 1 out of 1 series for tilt axis refinement
1/1, 00:00 remaining
Finished processing in 00:00:19
Current tilt axis angle: -90.802 °
Running AreTomo with final average tilt axis angle:
1/1, 00:00 remaining
Finished processing in 00:00:08
Saying goodbye to all workers... Done
(warptools) /data2A/mfrijns/CD25VCP/TestWarpTools$ module unload miniconda3/warptools/2.0.0
 
        Run "conda deactivate" to exit conda warptools environment
        Close the terminal to get rid of conda environment variables!
 

Unloading miniconda3/warptools/2.0.0
  Unloading useless requirement: cuda/toolkit/11.8
(warptools) /data2A/mfrijns/CD25VCP/TestWarpTools$ ml miniconda3/warptools/
 
        Run condaactivate to activate conda warptools environment
        Run condadeactivate or conda deactivate to exit conda environment
        Close the terminal to get rid of conda environment variables!
 

Loading miniconda3/warptools/2.0.0dev36
  Loading requirement: cuda/toolkit/11.8

(warptools) /data2A/mfrijns/CD25VCP/TestWarpTools$ condaactivate
(warptools_2.0.0dev36) /data2A/mfrijns/CD25VCP/TestWarpTools$ srun -n 1 --cpus-per-task 12 --partition=gpuq --gres=gpu:4 WarpTools  ts_aretomo --settings warp_tiltseries.settings --angpix 10 --axis_iter 1 --axis_batch 1 --exe /software/apps/aretomo/1.3.4/aretomo --alignz 1000
Running command ts_aretomo with:
angpix = 10
mask = False
alignz = 1000
axis_iter = 1
axis_batch = 1
min_fov = 0
axis = null
patches = null
delete_intermediate = False
thumbnails = False
exe = /software/apps/aretomo/1.3.4/aretomo
device_list = {  }
perdevice = 1
workers = {  }
settings = warp_tiltseries.settings
input_data = {  }
input_data_recursive = False
input_processing = null
output_processing = null
input_norawdata = False
strict = False

No alternative input specified, will use input parameters from warp_tiltseries.settings
File search will be relative to /data2A/mfrijns/CD25VCP/TestWarpTools/tomostar
1 files found
Parsing previous results for each item, if available...
1/1, previous metadata found for 0
Connecting to workers...
Connected to 1 workers
Current tilt axis angle: -88.700 °
Running iteration 1 of tilt axis refinement:
Using 1 out of 1 series for tilt axis refinement
1/1, 00:00 remaining
Finished processing in 00:00:21
Current tilt axis angle: -88.700 °
Running AreTomo with final average tilt axis angle:
1/1, 00:00 remaining
Finished processing in 00:00:06
Saying goodbye to all workers... Done

Op maandag 19 januari 2026 om 10:21:05 UTC+1 schreef Marten Chaillet:

[Marten Chaillet]

Hi Maurice,

I think you're right that it's not updated. I ran the AreTomo3 wrapper this morning with `--axis_iter 1` and it didn't update the tilt axis angle. When running with the etomo patch-tracking wrapper the tilt axis angle does update. 

Thanks for the comparison of 2.0.0  and 2.0.0dev36; definitely seems like something broke there for `ts_aretomo`, and `ts_aretomo3` likely has the same issue. 

Cheers,

Marten

Op maandag 19 januari 2026 om 12:43:04 UTC+1 schreef m.a.fri...@gmail.com: