Filament Reconstruction and Reference-based Boxing

[Liang Chen]

Dear EMAN2 Community,

I am trying to reconstruct actin filaments from a tomogram built by IMOD. Should I pick "particles" like the procedures described in EMAN2 tomography tutorial?

I used a hdf file (converted from pdb) as the reference for Reference-based Boxing, but the particles picked out do not look like the reference protein at all. Is it because I am using an IMOD built tomogram?

Best Regards,

Liang Chen

[Muyuan Chen]

From non-Eman tomograms, you can only save particles using the “save” button in the particle label widget of the manual particle boxer window.

References based picking should work on non-Eman tomograms, but there are parameters that can be incorrect if you import one from imod. Make sure the apix in header is correct and that the dark density in tomogram is protein while bright density in reference is protein. 

Of course if you can make a tomogram with Eman, debugging this would be easier...

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[Liang Chen]

I only found the apix in the "Import tiltseries". Since I imported tomograms from IMOD, where do I set the apix number in EMAN2? I tried to use EMAN2 to build my tomogram from tilt series and I followed the tutorial, but my particles are too blurry in the tomogram generated by EMAN2. I have not found a solution other than using IMOD.

Any suggestions about filament reconstruction?

Thank you very much for your help!

[Muyuan Chen]

You can do e2proc3d.py x.hdf x.hdf --apix x.xx to set apix

It is hard to give suggestions with the information I have. I do not know why your eman tomogram does not look good in the first place. There are programs in EMAN that does filament reconstruction but they wont work with non-eman tomograms.

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[Liang Chen]

Is it because my EMAN2 tomograms are binned? I can tell the filaments apart in the raw tilt series, but they all become blurry in the tomogram. I have not found any solutions for not to bin the tomogram.

By the way, is there any other EMAN2 tomography tutorial other than the brief official one?

[Muyuan Chen]

If your particles are only visible in fully sampled tomograms, you probably should consider increasing the magnification if you want to do averaging. You can actually set outsize to 4K to make full sized tomograms in Eman but it is not recommended.

There is only one tutorial for tomogram reconstruction, but there are a few other pages for other tomography related functions. You can search e2tomo in the eman2 wiki to find them.

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[Liang Chen]

Thank you for your help! I will keep learning EMAN2 to get a better understanding.