Preprocessing

At this moment, relion requires tomograms to be preprocessed externally. It is user responsability to perform the initial steps of movie frames alignment, tilt series alignment and particle picking.

In summary, the required information to start processing in relion is:

• Tilt series alignment: It should be done using IMOD package. Standard IMOD pipeline is typically accepted but note that not all IMOD options are supported.

• Initial CTF parameters: They can be estimated by either CTFFind or CtfPlotter.

• Order list: Chronological order of the tilt series acquisition.

• Fractional electron dose: electron dose per tilt image.

In this tutorial, we provide the files after computing these previous steps. For a full description of the preprocessing requirement, see relion_tomo_import_tomograms program.

Getting organised

We recommend to create a single directory per project, i.e. per structure you want to determine. We’ll call this the project directory. It is important to always launch the RELION graphical user-interface (GUI) from the project directory. Inside the project directory you should make a separate directory to store all your IMOD reconstructed tomograms folder. We like to call this directory tomograms/. Inside, we should find the different tomograms in folders like TS_1 or TS_01. If for some reason you do not want to place your tomograms inside the relion project directory, then inside the project directory you can also make a symbolic link to the directory where your reconstructed tomograms are stored.

relion uses the original tilt series stack prior to tilt axis alignment and imports the alignment data from IMOD. The tomogram folder should contain: a stack file, with extension .st, .mrc or mrcs; the newst.com and tilt.com script files, where the transformation and tilt angles filenames are obtained from together with tomogram dimensions, offsets and excluded views. Particle coordinates should refer to tomograms reconstructed using this information.

When you unpacked the tutorial test data, the tomograms/ directory was created. It should contain 5 tomogram folders. You should also find the masks/ and input/ folders. The last one should contains the files required to import tomograms data and coordinates.

We will start by launching the relion GUI. This GUI always needs to be launched from the project directory. To prevent errors with this, the GUI will ask for confirmation the first time you launch it in a new directory. Make sure you are inside the project directory, and launch the GUI by typing:

relion --tomo&

and answer Yes when prompted to set up a new relion project here.