Conventions & File formats

Image I/O

RELION reads the following image file formats:

• MRC individual images (with extension .mrc)
• MRC stacks (with extension .mrcs) (this is the recommended image format)
• SPIDER individual images (with extension .spi)
• SPIDER stacks (with extension .spi)
• IMAGIC stacks (with extensions .hed and .img)

Preparation of the images is explained on the Prepare_input_files page.

RELION writes individual images and image stacks in MRC format. Because there is no distinction between 3D maps or stacks of 2D images in the MRC format, 3D maps are indicated with a .mrc extension, and stacks with a .mrcs extension. Individual images in stacks are indicated by an integer number (ranging from 1 to the number of images in the stack) followed by an "@" sign and the filename of the stack. Thereby, the first three images in a stack called my_images.mrcs are called:

1@my_images.mrcs
2@my_images.mrcs
3@my_images.mrcs

Metadata I/O

The STAR format

RELION uses the STAR (Self-defining Text Archiving and Retrieval) format (Hall, Allen and Brown, 1991) for the storage of label-value pairs for all kinds of input and output metadata. The STAR format is an alternative to XML, but it is more readable and occupies less space. The STAR format has been adopted by the crystallographic community in the form of CIF (Crystallographic Information Framework), and Bernard Heymann's BSOFT package was the first to use STAR in the field of 3D-EM. Also Xmipp-3.0 now uses the STAR format.

RELION's implementation of the STAR format has the following rules (partially copied from BSOFT's manual):

• The file name must end in a ".star" extension.
• Each file must have one or more data blocks. The start of a data block is defined by the keyword "data_" followed by an optional string for identification (e.g., "data_images").
• Multiple values associated with one or more labels in a data block can be arranged in a table using the keyword "loop_" followed by the list of labels and columns of values. The values are delimited by whitespace (i.e., blanks, tabs, end-of-lines and carriage returns). The loop must be followed by an empty line to indicate its end.
• Label names always starts with an underscore ("_"). Each label may only be used once within each data block.
• Data items or values can be numeric or strings of characters. A string is interpreted as a single item when it doesn't contain spaces
• Comments are strings which can occur in three places:
  • File comments: All text before the first "data_" keyword
  • Data block comments: Strings on their own lines starting with "#" or with ";" as the first character in the line.
  • Item comments: Strings on the same line as and following tag-value items, also indicated by a leading "#".

Metadata label definitions

RELION has its own set of defined metadata labels. The command relion_refine --print_metadata_labels will print a list of the definitions of all of them.

An example

A STAR file that could be used as input for refinement in RELION that includes CTF information about each particle could look like this:

data_images
loop_
_rlnImageName
_rlnDefocusU
_rlnDefocusV
_rlnDefocusAngle
_rlnVoltage
_rlnAmplitudeContrast
_rlnSphericalAberration
000001@/lmb/home/scheres/data/VP7/all_images.mrcs 13538 13985 109.45 300 0.15 2
000002@/lmb/home/scheres/data/VP7/all_images.mrcs 13293 13796 109.45 300 0.15 2
000003@/lmb/home/scheres/data/VP7/all_images.mcrs 13626 14085 109.45 300 0.15 2

Orientations

Coordinate system

In compliance with the Heymann, Chagoyen and Belnap (2005) standard RELION uses a right-handed coordinate system with orthogonal axes X, Y and Z. Right-handed rotations are called positive.

Image center

The center of rotation of a 2D image of dimensions xdim x ydim is defined by ((int)xdim/2, (int)(ydim/2)) (with the first pixel in the upper left being (0,0). Note that for both xdim=ydim=65 and for xdim=ydim=64, the center will be at (32,32). This is the same convention as used in SPIDER and XMIPP. Origin offsets reported for individual images translate the image to its center and are to be applied BEFORE rotations.

Euler angle definitions

Euler angle definitions are according to the Heymann, Chagoyen and Belnap (2005) standard:

* The first rotation is denoted by phi or rot and is around the Z-axis.
* The second rotation is called theta or tilt and is around the new Y-axis.
* The third rotation is denoted by psi and is around the new Z axis

As such, RELION uses the same Euler angles as XMIPP, SPIDER and FREALIGN.

Contrast Transfer Function

CTF parameters are defined as in CTFFIND3, also see the publication by Mindell et al (2003)

Symmetry

Symmetry libraries have been copied from XMIPP. As such, with the exception of tetrahedral symmetry, they comply with the Heymann, Chagoyen and Belnap (2005) standard:

Symmetry Group	Notation	Origin	Orientation
Asymmetric	C1	User-defined	User-defined
Cyclic	C<n>	On symm axis, Z user-defined	Symm axis on Z
Dihedral	D<n>	Intersection of symm axes	principle symm axis on Z, 2-fold on X
Tetrahedral	T	Intersection of symm axes	3-fold axis on Z (deviating from Heymann et al!)
Octahedral	O	Intersection of symm axes	4-fold axes on X, Y, Z
Icosahedral	I<n>	Intersection of symm axes	++

++ Multiple settings of the icosahedral symmetry group have been implemented:

• I1: No-crowther 222 setting (=standard in Heymann et al): 2-fold axes on X,Y,Z. With the positive Z pointing at the viewer, the front-most 5-fold vertices are in YZ plane, and the front-most 3-fold axes are in the XZ plane.
• I2: Crowther 222 setting: 2-fold axes on X,Y,Z. With the positive Z pointing at the viewer, the front-most 5-fold vertices are in XZ plane, and the front-most 3-fold axes are in the YZ plane.
• I3: 52-setting (as in SPIDER?): 5-fold axis on Z and 2-fold on Y. With the positive Z pointing at the viewer and without taken into account the 5-fold vertex in Z, there is one of the front-most 5-fold vertices in -XZ plane
• I4: Alternative 52 setting: with the positive Z pointing at the viewer and without taken into account the 5-fold vertices in Z, there is one of the front-most 5-fold vertices in +XZ plane.

In case of doubt, a list of all employed symmetry operators may be printed to screen using the command (for example for the D7 group): reline_refine --sym D7 --print_symmetry_ops.