Prepare input files

Experimental images

RELION reads the following image file formats:

• MRC individual images (with extension .mrc)
• MRC stacks (with extension .mrcs) (recommended)
• SPIDER individual images (with extension .spi)
• SPIDER stacks (with extension .spi)

Preparation of the images is explained on the Preprocess images page. Further note that images should be squared (i.e. xdim=ydim.

If no CTF-correction is to be performed inside RELION, then a stack of images may be used directly as input (command line option --i). In that case, it is recommended that the images are CTF-phase flipped before refinement. If CTF-correction is to be performed inside RELION (recommended for cryo-data), then besides the images themselves, also metadata regarding the CTFs needs to be provided. In that case, the input to RELION is done using a STAR file (see below).

Metadata STAR files

The STAR file format is explained on the Conventions page. STAR files are easily readable plain text files, for which shell utilities like awk are very convenient. However, because not all users will be equally proficient in shell scripting, RELION comprises several shell script implementations to provide some basic operations with STAR files. See this page for a description of these utilities, of which relion_star_loopheader, relion_star_datablock_stack and relion_star_datablock_singlefiles are used below.

If ones' images are in a separate MRC stack for each micrograph (e.g. because they were picked using Jude Short's [Ximdisp program], then one could use the following commands to generate the input STAR file:

relion_star_loopheader rlnImageName rlnMicrographName rlnDefocusU rlnDefocusV rlnDefocusAngle rlnVoltage rlnSphericalAberration rlnAmplitudeContrast > my_images.star
relion_star_datablock_stack 4 mic1.mrcs mic1.mrcs 10000 10500 30 200 2 0.1  >> my_images.star
relion_star_datablock_stack 3 mic2.mrcs mic1.mrcs 21000 20500 25 200 2 0.1  >> my_images.star
relion_star_datablock_stack 2 mic1.mrcs mic1.mrcs 16000 15000 35 200 2 0.1  >> my_images.star

(Where the three stacks contain respectively, 4, 3 and 2 images.) This would result in this STAR file that could be used directly as input into RELION. Note the rlnMicrographName label, and the repetition of the micrograph names on the datablock lines, which will lead to the inclusion of a unique rlnMicrographName for each micrograph. By doing so, distinct noise spectra will be estimated for each micrograph.

Reference images

2D class averaging is typically performed in an unsupervised manner, i.e. without user-provided references. 3D reconstruction does require a (single) 3D reference structure. This map should be provided in MRC or SPIDER format, and it should have the same dimensions as the input images. Take care that the pixel size (in Angstroms) matches that of the experimental images, as currently an internal magnification correction is not implemented.