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.

Import coordinates

Ignore this part and refer to "manual picking" or "template-based semi-autopicking" if you decide to pick the helical tube/segment coordinates manually or automatically using Relion GUI. Picking coordinates is a personal experience. Relion supports helical tube/segment coordinates in EMAN2 (*.box) and XIMDISP (*.coords) formats as well. Relion prefers tube to segment coordinates because the former ones give users freedom to set the inter-box distance in particle extraction. Since the inter-box distance needs to be multiple of the helical rise in 3D reconstruction, please provide tube coordinates whenever possible, especially if the helical symmetry is not precisely clear before the start of the project.

Tube coordinate files in EMAN2 (*.box) format should contain exactly the following content (ignore comments after // symbol):

1463    3307    260     260     -1        // Tube 1 starting coordinates: x, y, box width, box width, -1 
851     2211    260     260     -2        // Tube 1 end coordinates: x, y, box width, box width, -2 
407     2039    260     260     -1        // Tube 2 
-45     1482    260     260     -2 
... 

EMAN2 box widths are ignored and overwritten by the box size specified in particle extraction step.

Tube coordinate files in XIMDISP (*.coords) format should contain exactly the following content (ignore comments after // symbol):

Box     1 
         750         670    // Top-left coordinates of rubberband box 1
        1245         375    // Top-right coordinates of rubberband box 1
        2980        3275    // Bottom-left coordinates of rubberband box 1
        2485        3570    // Bottom-right coordinates of rubberband box 1
         750         670    // Top-left coordinates of rubberband box 1
Box     2 
        2500        3575 
        1925        3560 
        1990         515 
        2565         530 
        2500        3575 
... 

Tube coordinate files in Relion (*.star) format should at least contain the x, y coordinates for each filament (ignore comments after // symbol):

data_ 

loop_ 
_rlnCoordinateX #1 
_rlnCoordinateY #2 
  110.000000  1080.000000   // Tube 1 starting coordinates x, y 
 1855.000000   585.000000   // Tube 1 end coordinates x, y 
  635.000000  1325.000000   // Tube 2 
  560.000000  2490.000000 
... 

Segment coordinate files in XIMDISP (*.coords) format should contain exactly the following content (ignore comments after // symbol):

  x    y    psi           // One-line header 
1043    3380    7.125     // Segment 1: x, y coordinates, in-plane rotation angle (in degrees) 
1019    3383    7.125     // Segment 2
995     3386    7.125 
970     3389    7.125 
946     3392    7.125 
...

In-plane rotation angles are transformed according to Relion convention in particle extraction.

Segment coordinate files in RELION (*.star) format should at least contain the following prior information for each segment:

data_ 

loop_ 
_rlnCoordinateX #1 
_rlnCoordinateY #2 
_rlnHelicalTubeID #3 
_rlnAngleTiltPrior #4 
_rlnAnglePsiPrior #5 
_rlnHelicalTrackLength #6 
_rlnAnglePsiFlipRatio #7 
 1822.915020   227.604136            1    90.000000   -58.642915    43.599998     0.500000 
 1845.603159   264.835953            1    90.000000   -58.642915    87.199997     0.500000 
 1868.291298   302.067770            1    90.000000   -58.642915   130.799995     0.500000 
 1890.979436   339.299588            1    90.000000   -58.642915   174.399994     0.500000 
 1913.667575   376.531405            1    90.000000   -58.642915   217.999992     0.500000 
...

The ways to import movies/micrographs and coordinate files are explained in the Relion 2.0 tutorial. Please note that no errors are raised if you have accidentally made mistakes in handling imports until particle extraction. Please make sure that the file contents and the filenames with wildcards (*, ?) are provided correctly. In addition, Relion requires that the movie/micrograph file exists if the coordinate file with the same rootname is imported.

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