Helical Image Processing (PHOELIX)¶

General Workflow:¶

1. Make sure that the appropriate run name and directory tree are specified. Appion increments names automatically, but users are free to specify proprietary names and directories.
2. Enter the directory tree where the mandatory files are located. These files include llbo.sa, strong.sa, range.sa, cutfit1.dek, cutfit2.dek, cutfit3.dek, chop1.dek, chop2.dek, and template. If you do not have these files already, run preHIP as described in the next section and follow the guided protocol to generate them.
3. Enter a description of your run into the description box.
4. Select the stack to process from the drop down menu. Note that stacks can be identified in this menu by stack name, stack ID, and that the number of particles, pixel and box sizes are listed for each.
5. Make sure that "Commit to Database" box is checked. (For test runs in which you do not wish to store results in the database this box can be unchecked. This box is automatically deactivated when running preHIP).
6. Enter the Filament Parameters or verify the default values.
   • Number of Filament Segments - The number of particles to use in the run.
   • Repeat Length - (Helical Repeat) The 360 degree repeat distance along the filament, in Ångströms. Calculated in Fourier space by the distance to the first layer line or in real space by the distance it takes one subunit to travel 360°.
   • Subunits per Repeat - Number of subunits in one helical repeat. Determined by calculating (Helical Repeat/Rise) or finding the Layer Line with a J0 intercept.
   • Diameter - The approximate outer diameter of the filament, in Ångströms
   • Inner Diameter - The approximate inner diameter of the filament, in Ångströms.
7. Enter the Processing Parameters or verify the default values.
   • Filament Segment Length - Desired length of the filament segments, usually a certain number of helical repeats, in pixels. Should be the same as your stack box size.
   • Box Height - Height of the filament box after alignments, usually the power of 2 value greater than the filament diameter, in pixels.
   • Pad Value - Value to pad the filament box to during alignments, usually the power of 2 value greater than the filament length/box size.
   • Phase Residual Cutoff - Maximum phase residual for post averaging. A phase residual is calculated between the layer lines from the template and the layer line from each filament segment. Any files with phase residual above this cutoff will not be included in the final average, map, overplot, etc.
   • Filter Value - Lowpass filter value for filament segments used during alignments to find filament edges. 150-200 seems to work well, but should be tested using preHIP before processing the full stack
   • Binning - Binning of the particle images. This quickly shrinks the image to help make the processing faster.
   • Contrast Change - If your filament stack has black on white density, the final reconstruction will need a contrast change.
8. Click on "Run HIP" to submit your job to the cluster. Alternatively, click on "Just Show Command" to obtain a command that can be pasted into a UNIX shell.

Running PreHIP:¶

1. Click the "Run PreHIP" checkbox which will activate the indexing parameters below it and display a warning message.
2. Fill in the Indexing Parameters.
   • N-fold - Order of the symmetry axis. Default is 1-fold for no symmetry.
   • Maximum Layer Line - Maximum layer line to include in the llbo.sa file.
   • Maximum Bessel Order - Maximum Bessel Order to include in the computation for llbo.sa.
3. Select either the "Use rise and twist" or "Use layer line/bessel order" radio button which will activate the parameters below it. You can only choose one of these options, therefore the one not selected becomes deactivated.
   • Rise and Twist - Use the rise distance and twist angle between protein subunits to determine selection rule and generate llbo.sa file. Rise is the vertical distance between two subunits in the one-start helix and twist is the angle between two adjacent subunits.
   • (1,0),(0,1) LLBO - Use the layer line and Bessel Order for the (1,0) and (0,1) layer lines to determine selection rule and generate llbo.sa file.
4. Click "Just Show Command" to generate the UNIX shell command.
5. Note that the "Commit to Database" box is deactivated. preHIP is used only for generating the mandatory files and therefore the preliminary results are not stored to the database.