Cannot obtain correct apoferritin STA reconstruction following WarpTools tutorial
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rui wang
Jun 16, 2026, 7:50:17 AMJun 16
to Warp
Hello, I am trying to reproduce the WarpTools(version 2.0.0) cryo-ET tutorial using the provided apoferritin dataset(EMPIAR-10491) and followed the tutorial workflow in WarpTools. The entire processing pipeline in WarpTools completed successfully without any obvious errors, including motion correction, CTF estimation, tilt-series alignment(Etomo), tomogram reconstruction, particle picking(WarpTools TM), and export(3d subtomo) to RELION4. However, after performing subtomogram averaging and refinement in RELION, I am unable to obtain the expected apoferritin structure. The tomograms appear reasonable, and I have not found any obvious problems in the processing logs. Has anyone encountered a similar issue or could suggest what I should check first?
For reference, I have attached representative images of the reconstructed tomograms, extracted particles, and the final STA map obtained from RELION. I am relatively new to cryo-ET and still learning the workflow, so I would greatly appreciate any suggestions or guidance from the community. Thank you very much for your time and help.
For reference, I have attached representative images of the reconstructed tomograms, extracted particles, and the final STA map obtained from RELION. I am relatively new to cryo-ET and still learning the workflow, so I would greatly appreciate any suggestions or guidance from the community. Thank you very much for your time and help.
Warp Bot
Jul 9, 2026, 9:03:51 PM (5 days ago) Jul 9
to Warp, rui wang
Hello,
A few things to check before diving deeper:
1. Export mode and RELION version mismatch. You mention exporting "3d subtomo" to RELION4. The current WarpTools tutorial (https://warpem.github.io/user_guide/warptools/quick_start_warptools_tilt_series/) uses 2D particle series exported via ts_export_particles --2d, targeting RELION-5 (not RELION4). If you followed an older tutorial or used --3d instead of --2d, that is the most likely explanation for poor results. The two workflows are not interchangeable.
2. What exact ts_export_particles command did you run? Specifically:
- Did you use --2d or --3d?
- What --output_angpix and --box values?
- Did you use --normalized_coords (required if coordinates came from WarpTools template matching)?
3. Which RELION version are you using? For the 2D particle series workflow you need RELION-5 and should start refinement from the relion --tomo GUI (not the standard GUI). If using 3D subtomograms, refinement goes through the standard relion GUI, NOT relion --tomo.
If you can share the ts_export_particles command and the RELION version and job type used for refinement, that will narrow this down quickly.
— 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.
A few things to check before diving deeper:
1. Export mode and RELION version mismatch. You mention exporting "3d subtomo" to RELION4. The current WarpTools tutorial (https://warpem.github.io/user_guide/warptools/quick_start_warptools_tilt_series/) uses 2D particle series exported via ts_export_particles --2d, targeting RELION-5 (not RELION4). If you followed an older tutorial or used --3d instead of --2d, that is the most likely explanation for poor results. The two workflows are not interchangeable.
2. What exact ts_export_particles command did you run? Specifically:
- Did you use --2d or --3d?
- What --output_angpix and --box values?
- Did you use --normalized_coords (required if coordinates came from WarpTools template matching)?
3. Which RELION version are you using? For the 2D particle series workflow you need RELION-5 and should start refinement from the relion --tomo GUI (not the standard GUI). If using 3D subtomograms, refinement goes through the standard relion GUI, NOT relion --tomo.
If you can share the ts_export_particles command and the RELION version and job type used for refinement, that will narrow this down quickly.
— 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.
On Tue, Jun 16, 2026 at 11:50 AM UTC rui wang <wang...@gmail.com> wrote:
Hello, I am trying to reproduce the WarpTools(version 2.0.0) cryo-ET tutorial using the provided apoferritin dataset(EMPIAR-10491) and followed the tutorial workflow in WarpTools. The entire processing pipeline in WarpTools completed successfully without any obvious errors, including motion correction, CTF estimation, tilt-series alignment(Etomo), tomogram reconstruction, particle picking(WarpTools TM), and export(3d subtomo) to RELION4. However, after performing subtomogram averaging and refinement in RELION, I am unable to obtain the expected apoferritin structure. The tomograms appear reasonable, and I have not found any obvious problems in the processing logs. Has anyone encountered a similar issue or could suggest what I should check first?
For reference, I have attached representative images of the reconstructed tomograms, extracted particles, and the final STA map obtained from RELION. I am relatively new to cryo-ET and still learning the workflow, so I would greatly appreciate any suggestions or guidance from the community. Thank you very much for your time and help.![p2.png]()
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