Process movies

As of version 1.2, RELION incorporates functionality to process movies from fast digital cameras, such as direct-electron detection devices (DDDs). The following has been tested on several data sets from our FEI Falcon camera. Other detectors have not been tested yet. Please share your experiences with us if you have.

Get organised

Organize all your movies and the average micrographs (i.e. single-image files that are the average of all frames of your individual movies) inside one or more directories that are inside your RELION project directory (from where you would launch the GUI). We like to call these directories "Micrographs/" if all micrographs are in one directory, or "Micrographs_15jan13/" and "Micrographs_23jan13/" if they are in different directories (e.g. because they were collected on different dates. But, you can organise yourself as you like.

Name the average micrographs and movies in the following way:

001.mrc
001_movie.mrcs
24.01.13.15.04.mrc
24.01.13.15.04_movie.mrcs
myverybest.mrc
myverybest_movie.mrcs
...

Note that all average micrographs are in MRC format with an .mrc extension, and all movies are MRC stacks with a .mrcs extension. It is important that movies have the same rootname (e.g. 001, or 24.01.13.15.04, or whatever you like) as their corresponding average micrographs, plus a movie-identifier (in this case "movie") that follows the rootname after an additional "_" character.

What to do first

First, use the average micrographs to extract "normal" particle stacks, and follow the Recommended_procedures for 3D refinement.

Preprocessing movies

Then, extract (box out) the individual movie-frames of all particles from the original micrograph movies. To do this, go back to the Preprocessing run-type in the RELION GUI, and:

  * Keep everything on the I/O tab as it was when you extracted the "normal" particles   (also do NOT change the rootname)
  * Set "Run Ctffind3?" to No
  * Set "Extract particles from micrographs?" to Yes
  * Set "Use movies instead of micrographs?" to Yes
  * Set "Rootname of movie files" to the movie-identifier mentioned above (in the example above "movie")
  * Set "Number of frames to average?" to any desired number from 1 to half the number of frames in your movies
  * Keep everything on the operate tab as it was when you extracted the "normal" particles   
  * Let it run... !

(By this time you will probably already be aware of disc quota, full discs, etc....) For each movie, a separate stack with all movie frames of all particles in that micrograph will be stored along the extracted "normal" particle stacks in the Particles directory. All metadata regarding these particle-movies will be stored in a large STAR file in your project directory that is called particles_movie.star (where "particles" is the Particle rootname given on the I/O tab; and "movie" is your movie-identifier).

3D refinement with movies

Finally, go to the 3D auto-refine run-type in the GUI and load the settings of the run that you want to perform movie-processing on. Note that this previous 3D auto-refine run was done with (most likely a classified subset of) the average-micrographs particles. As long as the naming conventions outlines above are maintained, then the program will figure out itself which movie particles correspond to which average-particle in the 3D auto-refine. In the 3D auto-refine GUI:

  * Select "continue old run" from the pull-down menu on the top.
  * Possibly change the "Output rootname" (also see below)
  * Select the last optimiser.star file from the run at the "Continue from here" option
  * Under the Optimisation tab, set "Realign movie frames?" to Yes.
  * Set "Input movie frames" to the particles_movie.star file generated in the section above.
  * Select suitable parameters for "Running avg window" and the standard deviations of the priors on the rotations and translations. 
  * If you plan to use these movies for subsequent particle polishing (see below), then you can skip the rotational searches
  * Keep everything on the CTF and Sampling tabs unchanged
  * Let it run... !

Regarding the parameter choices on the Movie tab: the defaults worked well for our ribosome data, but this will depend strongly on your total dose, how much your particles move etc. One can run this procedure multiple times (if you change the "Output rootname" on the I/O tab, previous results will not be overwritten). You can then select optimal parameter settings based on the gold-standard FSC curve.

Post-processing

Whether you only perform the original movie-processing refinement, or you also perform particle polishing, remember to always get the most out of your final reconstruction by calculating mask-corrected FSCs and sharpening your map, as implemented in the postprocessing job-type, and as explained on the Analyse results page.