Helical processing

Our results

A paper about helical processing in Relion 2.0 is still in preparation.

Usage of relion_helix_toolbox

Relion_helix_toolbox is a standalone executable in Relion 2.0 and features many useful tools which we often use when processing helical data. The command relion_helix_toolbox (without any options) displays the full list of functions and parameters available in the error message. Type the command relion_helix_toolbox [function] --help for the usage of a function. We have implemented tools such as reference building (--cylinder, --pdb_helix, --simulate_segments), masking (--spherical_mask), imposition and local searches of helical twist and rise (--impose, --search), bad segment removal ("--remove_bad_tilt", "--remove_bad_psi"), splitting and merging of STAR files (--divide, --merge), etc.

Mask creation

The top and bottom parts of the reconstructed helical map suffer from inaccuracies of orientations. A mask which only covers the central part might improve the overall resolution. After finding out the best values for parameters on "Mask" tab, we usually set the central Z length to 30% and increase it gradually at steps of 5% to find the largest value (<=80%, because further parts are masked out anyway) which still gives the best estimated resolution. Also it seems that wider soft edges (5~8 pixels) are useful for helices.

Particle polishing

Enable helical reconstruction and copy the number of asymmetrical units from the previous 3D refinement job. Copy the helical symmetry as well if local searches of helical symmetry have not been performed. Otherwise look into the file Refine3D/jobXXX/run_model.star for the averaged helical symmetry at the end of the refinement:

... 
data_model_classes 

loop_ 
_rlnReferenceImage #1 
_rlnClassDistribution #2 
_rlnAccuracyRotations #3 
_rlnAccuracyTranslations #4 
_rlnHelicalRise #5 
_rlnHelicalTwist #6 
Refine3D/jobXXX/run_class001.mrc     1.000000     0.186000     0.250000    21.775977    29.410791 
...

References

We gain experience for helical processing in Relion using the movies and micrographs from EMDB's EMPIAR database (accession number EMPIAR-10019, 10020, 10021, 10022, 10031). The related research papers are listed below:

[1] Kudryashev, Mikhail, et al. "Structure of the type VI secretion system contractile sheath." Cell 160.5 (2015): 952-962. PubMed

[2] Fromm, Simon A., et al. "Seeing tobacco mosaic virus through direct electron detectors." Journal of structural biology 189.2 (2015): 87-97. PubMed

[3] Xu, Hui, et al. "Correction: Structural basis for the prion-like MAVS filaments in antiviral innate immunity." Elife 4 (2015): e07546. PubMed