bad alignments on automated tomogram reconstruction

[Simona Sorrentino]

Hello,

 I acquired some tomograms for investigating particles distribution (the usual question: are they at the air water interface?). The protein is a small protein, which gives really bad contrast, so I used a mag with 1.1 A/pixel, acquired on Titan Krios G3i, 300 KV. I acquired some of the tomograms on position with ice contamination, hoping that it would help the alignemt (picture attached). The stage was super stable, so the tilt series is really not moving but the reconstruction was awful (also, pic attached).

The eman version I used is 2.9.

I would take any suggestion for next time, (e.g. which pixel size you would use) and or any tricks to obtain the reconstruction from this tomogram.

Thanks in advance

Simona

[Muyuan Chen]

If you are willing to send a sample dataset privately, I can look into this and try to figure out what’s wrong.

Right now, my general suggestions are: 

did you provide the correct tilt axis angle? Sometimes the default tilt axis estimation can be off by a lot when there isn’t a lot of low resolution signal within the sample. Also make sure the tilt step is correct too.

If you update to 2.91, try adding the —patchtrack=1 option, which may help when there are few landmarks.

Muyuan 

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<Tomo_BadAlignment.PNG>

<TiltExample.PNG>

[Simona Sorrentino]

Dear Muyan,

 yes, I am willing to send a dataset.

In the meantime, I tried with your suggestions:

the tilt step was correct, but I did not specify the tilt angles (I only used eman2 for real tomography data, with more features and fiducials, so it always worked without specifying it).

So, after trying with the tilt angle specification, and the —patchtrack=1 option things improved.

Attached there is an image of the ice contamination, better aligned then before, but not perfect yet.

best

Simona

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