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.
Particle polishing
For relatively small particles (e.g. sub-MegaDalton), following beam-induced movements by aligning particles in running averages of a few movie frames may become very noisy. To make this procedure more robust, a so-called particle-polishing procedure was implemented in RELION-1.3. It improves movie-refinement in two ways:
- Linear movement tracks are fitted through the (possible very noisy) movement tracks as determined by the original movie-refinement (as outlined above). To further increase robustness to noisy tracks, multiple neighbouring particles on the same micrograph are considered simultaneously in this fitting. This reduces noise, since neighbouring particles are often observed to move in similar directions. To still be able to model complicated movement patterns, where particles in different areas of the micrograph move in different directions, the user defines a standard deviation of the particle-distance, which is used to calculate a Gaussian weight on the least-squares fit of these multiple particles. Thereby, particles far away from each other contribute little to each others fitted tracks, whereas particles within a 1-2 standard deviations still contribute significantly to each others fitted tracks.
- A resolution and dose-dependent weighting scheme is devised to model unresolved beam-induced movements and radiation damage. Radiation damage affects the high-resolution components at much lower electron dose than the low-resolution components. Consequently, whereas the high-resolution signal in the later frames may be completely gone, these frames may still contribute positively to the low-resolution signal (and thereby the orientability of the particles). The resolution-dependent decay of each movie frame is modelled by a B-factor, which is estimated from the gold-standard FSC between individual movie-frame reconstructions. These FSCs are converted into relative Guinier plots, which are the logarithm of the amplitudes of the individual-frame reconstructions divided by the amplitudes of the average reconstruction from all frames versus the square of the resolution. Linear fits through these Guinier plots (which may often be performed for resolution higher than 20 Angstroms) then yield a slop (the B-factor) and an intercept (a resolution-independent scale-factor) which are used to device the resolution-dependent weghting scheme. Often, the B-factors are relatively large during the first few electrons/squared Angstroms dose due to unresolved, large initial beam-induced movements, and then also become relatively large again in the later frames when radiation damage sets in. A similar behaviour is also often seen for the intercepts, although sometimes for those the initial frames are as good as the middle ones.
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.