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Extracting particles using Relion

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Michael A. Cianfrocco edited this page Jun 9, 2018 · 2 revisions

Home > Extracting particles > Extracting particles using Relion

Extracting particles using Relion

To read about the overall workflow for using Relion, see here.

  1. Preparing inputs for Relion
  2. Launching & utilizing Relion's GUI
  3. Executing particle extraction command

Preparing inputs for Relion

For CTF-corrected data analysis, Relion expects the following inputs:

  • _ctffind3.log files for each micrograph (output by estimateCTF_CTFFIND3.py)
  • A star file containing per-micrograph CTF parameters (e.g. all_micrographs.star,output by estimateCTF_CTFFIND3.py)
  • .box files for each micrograph (output by automated/manual particle picking)
  • .mrc micrograph files

For non-CTF corrected data analysis, Relion expects the following inputs:

  • A star file containing a list of micrographs (e.g. all_micrographs.star,output by estimateCTF_CTFFIND3.py)
  • .box files for each micrograph (output by automated/manual particle picking)
  • .mrc micrograph files

For instance, if you were going to extract particles using Relion with CTF correction: 1) You would have a text file all_micrographs.star in your current working directory, and 2) you would have the following files in a folder named 'Micrographs':

$ ls Micrographs/
100_movie_gc.box
100_movie_gc_ctffind3.log
100_movie_gc.mrc
101_movie_gc.box
101_movie_gc_ctffind3.log
101_movie_gc.mrc
...

Launching & utilizing Relion's GUI

Relion provides a GUI to guide users through the input options required for all steps. To launch the GUI and put it in the background, type:

$ relion &

This will open a new window, where you need to input the DETECTOR PIXEL SIZE where it says 'Magnified pixel size (Angstrom)' along with the diameter of your particles in Angstroms.

How to calculate detector pixel size:

Pixel size = (Physical pixel size of detector)/(Magnification)

Now click on 'Particle extract' within the left menu.

This will show a series of inputs required:

  • The per-micrograph CTF information file (e.g. all_micrographs_ctf.star)
  • Extension of particles coordinates (.box)
  • Output rootname for particle stacks, info files, and output .star files (particles)

Now click on the next tab within this menu ('extract') and you will need to consider the following options:

  • Extract particles from micrographs: YES
  • Box size of extracted particles in pixels from the unbinned micrograph (read here for a discussion of box size)
  • Invert contrast: YES invert if cryo-EM data, NO if negative stain
    • Particles need to 'white' on 'black/grey' background
  • If you want to bin your data to speed up processing, scale the box size from the box size of extracted particle to a new size for binning.
    • In the example, the original box size of 240x240 pixels will be scaled to 120x120, which is binning the data by 2
  • Normalize particles - YES.
    • Provide values of 4 or 5 here. This means that any pixels that are +/-5 standard deviations away from the average pixel value will be discarded and replaced by a random value

For now, we will skip the 'movies' option and we don't need Relion to help use with our MPI thread number.

So, when finished with the binning and normalization, click 'Print Command'

This will output a command like to the command line:

*** The command is:
`which relion_preprocess` --o particles --mic_star all_micrographs_ctf.star --coord_suffix .box --extract --extract_size 240 --scale 120 --norm --bg_radius 4 --white_dust 5 --black_dust 5 --invert_contrast

Executing particle extraction command

With this command that was output by Relion, it is a good idea to record this command in a text file somewhere so t you remember what you did to extract your particles.

Fortunately, Relion offers users the ability to extract particles in a multi-processor fashion, which means that if your workstation has more than one processor (e.g. 8), then you can run a command that looks like this:

$ mpirun -np 8 relion_preprocess_mpi -o particles --mic_star all_micrographs_ctf.star --coord_suffix .box --extract --extract_size 240 --scale 120 --norm --bg_radius 4 --white_dust 5 --black_dust 5 --invert_contrast

The output particles and .star files will be put into the folder:

Particles/Micrographs

Where each particle stack (.mrcs) will be associated with an information file (.star), named:

[micro name][rootname].mrcs / .star

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