Warptools reconstruction and denoise

[Zhu Qiao]

Hi community

I am very new to the tomography and subtomogram averaging. Really appreciate it if I can get helps/suggestions about my tomo data processing.

When I collected the tilt series data, I used the on the fly ARETomo3 data processing pipline. and it generated super good contrast tomograms with bin8. 

I also processed the raw data in Warptools and imported the ARETomo3 alignment settings. But, then the reconstructed tomogram looks not as good as ARETomo3, visually. I also noticed the image plan shifted/rotated a bit. My question is how can I get the tomogram without the image rotation? Please see the pictures attached. 

I tried the Noise2map in Warp and denoised my tomogram, but looks like no difference. what parameters shall I adjust to make the constrast better? Becuase I use the current tomograms, the template matching results are not very good. I am thinking denosieng the tomogram could help template matching. 

here is the commands  for denoise (Noise2Map --a warp_tiltseries/reconstruction/even --b warp_tiltseries/reconstruction/odd --observation_combined warp_tiltseries/reconstruction --iterations 600 --angpix 10 --dont_flatten_spectrum)

Best Regards

Zhu 

[Daniel Cai]

Hi Zhu,

My experience regarding Warp vs Aretomo reconstruction readability is similar to yours. Not sure if you've tried inverting the warp reconstruction contrast in 3dmod, but that helps me see better. That being said, the approaches that Warp and Aretomo use to generate the tomograms and correct CTF are different, and I think this difference in human readability is to be expected:

My 10000 foot understanding is that Aretomo's SIRT and other program's SART iterative algorithms result in good high-contrast human readable data while also sacrificing some high frequency signal.

On the other hand, Warp's 3d fourier reconstruction and other softwares (like IMOD's) weighted back projection, should retain the high frequency signal, but would be more noisy to the human eye. 

I think that the image plane shift is probably a result of the tilt-axis not being at 90 degrees (most krios seem to be around ~85-86). My experience is that most software, including previous versions of AreTomo if I remember correctly, have that plane shift, and thus I'm not sure how correctable it is. 

Regarding noise2noise, does 10Å/pix correspond to your desired bin8? Also, while by eye the difference might not be big, it's possible that a computer likes the data much more. For template matching in Warp, make sure the template particle is scaled correctly to the pixel size and test different box sizes suitable for your particle. When I first tried template matching I accidentally scaled the particle way too small and was very confused with all the false positives! If you have time, I also think it's a good idea to compare template matching results with your Aretomo3 reconstruction along with different denoising pipelines like cryocare-IsoNet.

Adding onto this, the Noise2noise might also be cleaning up the data in ways that are more computer-friendly than human readable. Also, does 10Å/pix correspond with your bin8? If you are still having problems with the template matching have you tried other denoising pipelines like cryocare-isonet or deepdewedge?

Sorry if this wasn't a satisfying answer - information on cryo-ET processing can still be opaque and not easy to find. 

Good luck,

Daniel

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[Alister Burt]

Hi Zhu,

Warp tomograms are not filtered to make them ideal for visualization, they are supposed to keep high resolution information intact for things like template matching.

If you need extra contrast you can try adding the --deconv flag to filter the reconstructions or you can denoise using Noise2Map

The empty regions you see in your tomogram are regions where not all tilts contribute to the tomogram, you can try playing with the --keep_full_voxels flag but beware there are some interactions with template matching

Denoising will not help your template matching, just because tomograms look visually better to you does not mean the information is more similar to a given template, our eyes like low frequency information

I'm not sure why your noise2map runs are not working, try to work with one tomogram at a time and play with the available parameters - number of iterations is the main one.

Cheers,

Alister

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[Zhu Qiao]

Dear Alister

Thanks for your reply. I will play around following your suggestions.

Regards

Zhu