refinement of two different filaments

[Laurence serre]

Dear Eman2 developers

version: Eman 2.99.47

I used subtomogram averaging method to solve a model which consists in two species of filaments (A and B-filament). I would like to determine (if possible) the relative polarity of one with respect to the other. The two filaments cannot be refined together as they have different periodicity.

1.     -I picked up the subtomograms using Eman2’s filament option and extract the particles

2.     -I solved and orientated the thichker filament (A-filament) with “ e2spt_refine_new.py” and obtain a good model at 9.0 A resolution using a known model of A-filament.

e2spt_refine_new.py --ptcls=sets/particles.lst --ref=A-filament.hdf --startres=50.0 --goldstandard --sym=c1 --iters=p3,t2,p,t,r,d --keep=0.95 --maxres=0.0 --minres=0.0 --parallel=thread:22 --threads=20 --curve

 

3.     -To solve the B-filament, I used ptcls=spt_01/aliptcls3d_06.lst as input and a mask defined around B-filament location and a known model of B-filament. Final model is about 9.0 A resolution.

e2spt_refine_new.py --ptcls=spt_01/aliptcls3d_06.lst --ref=B-filament.hdf --startres=50.0 --goldstandard --sym=c1 --iters=p3,t2,p,t,r,d --keep=0.95 --loadali2d=spt_01/aliptcls2d_06.lst --maxres=0.0 --minres=0.0 --parallel=thread:28 --threads=24 --curve --mask=mask-B-filament.hdf

 

4.     -I checked the polarity of the final refined B-filament and it always matches the polarity of the input reference (I used both polarities for B-filament).

Therefore, it seems that the initial orientation parameters from spt_01/aliptcls3d_06.lst are not considered to orient the B-filament.

 

My question is: Is there a way to prevent the flipping of the aligned particles of the A-filament ? 

…and to determine whether there is or there isn’t a specific polarity of the B-filament relatively to the A-filament ?

 

I am not sure this is really clear…sorry. Thanks in advance for your help.

Laurence Serre, Grenoble Institute of Neuosciences, FRANCE

[Steve Ludtke]

EMAN2 has moved away from "stable versions" to rolling releases with distribution via conda-forge. Your version is ~4 years old, so there is a good chance that the problem has been fixed. Regardless, the first thing to try is to get a current release with the current installation strategy and see if the problem still occurs.

https://blake.bcm.edu/emanwiki/doku.php?id=eman2:install:condainstall

Note that there is still a choice between installing the last compiled binary or installing from source, which gives the flexibility of picking any specific GitHub time-point. Binaries are generated only when one of the devs thinks they have done something important. Minor changes and bugfixes hang around in github until that time.

Actually, it looks like it's been a while, so maybe time to push a new binary to catch all of the smaller changes over the last few months.

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[Muyuan Chen]

If you want to preserve the alignment of spt_01/aliptcls3d_06.lst, use --localrefine instead of --curve. 

You may find some useful information in the tutorial. 

https://blake.bcm.edu/emanwiki/doku.php?id=eman2:e2tomo_p22

It is for viruses but it includes discussion on how to refine different parts of the same protein with different symmetry. 

If the A filament refinement looks good, and you can see some density of B filament from the A filament structure, maybe it is possible to get the B filament structure from the particles themselves, similar to the virus tail finding, so it avoids the bias from the reference. But if you have to use a reference, and you are using --localrefine, make sure to move the reference to the roughly B filament location in the A filament average before the refinement. Otherwise it would just align the A filament density to the B filament reference. 

If you want to determine if there is a specific polarity, consider adding --breaksym=d1 in addition to --localrefine. But for that you will need to re-extract particles with shifted coordinates first so the B filament is roughly along the z axis. 

Muyuan

[Laurence serre]

thanks a lot for your suggestions..

L.