Using Relion with SPIDER data

Overview

According to Sjors Scheres at the MRC Laboratory of Molecular Biology:

RELION (for REgularised LIkelihood OptimisatioN) is a stand-alone computer program for Maximum A Posteriori (MAP) refinement of (multiple) 3D reconstructions or 2D class averages in electron cryo-microscopy (cryo-EM). It is developed in the group of Sjors Scheres at the MRC Laboratory of Molecular Biology. Briefly, the ill-posed problem of 3D-reconstruction is regularised by incorporating prior knowledge: the fact that macromolecular structures are smooth, i.e. they have limited power in the Fourier domain. In the corresponding Bayesian framework, many parameters of a statistical model are learned from the data, which leads to objective and high-quality results without the need for user expertise. the underlying theory of MAP refinement is given in Scheres (2012) JMB. A more detailed description of its implementation is given in Scheres (2012) JSB.


Usage

  1. Create/select a low pass filtered reference volume of same size and scaling as your particle data.

    Example:
    ¤ mkdir -p relion/input ; cd relion    Create and access input directory
    ¤ cp myproject/Reconstruction/refvol.dat   input    Get reference volume (one)

  2. Create/select a group selection file, particle selection file, and particle stacks and place in input directory. Note that particle image files must be square and even-dimensioned.

    input/sel_group Group selection doc file with group number, number of particles, and group defocus
    input/sel_particles Particle selection doc files (one/group)
    input/data*** Unaligned particle stacks (one/group)
    Example:
    ¤ cp myproject/Alignment/sel_group.fmr input    Get group selection doc file (one)
    ¤ cp myproject/Alignment/sel_particles_* input    Get particle selection doc files
    ¤ cp myproject/Alignment/data* input    Get Unaligned particle stacks
    ¤ cp myproject/params.* .    Get microscpy parameters file for reference

  3. Normalize groups of SPIDER experimental image stacks and create 'star' input file for Relion. The normalized particles will have average density of zero in the background with a standard deviation in the noise of one.

    Adjust imageing parameters and filenames in SPIDER procedure: norm_n_star.spi which reads:
    sel_group Group selection doc file with group number, number of particles, and group defocus
    Alignment/sel_particles Particle selection doc files (one/group)
    Alignment/data*** Unaligned particle stacks (one/group)
    and creates:
    ¤ input/relion_[id]_data.star Relion star file (one)
    ¤ input/data_norm_*** Normalized particle stacks (one / group)

    Example:

    ¤ spider spi/fmr @norm_n_star    Normalize images and create Relion star file

  4. Run Relion on compute cluster. Refer to SPIDER params.dat file for microscopy imaging parameters. Example:

    ¤ relion` Starts Relion GUI
    Run type: 3D classification
    Start new run: Yes
    I/O Input images: input/relion_1_data.star
    I/O Output rootname: run1/relion
    I/O Number of classes: 4
    I/O Reference map: input/refvol.dat
    I/O Reference map absolute greyscale: No
    I/O Symmetry (for ribosome): C 1
    CTF Pixel size: 2.78
    CTF Do CTF-correction: Yes
    CTF Has reference been CTF-corrected: No
    CTF Have data been phase flipped: No
    CTF Ignore CTFs until first peak: No
    Optimisation Initial low-pass filter A: 60
    Optimisation Number of iterations: 25
    Optimisation Regularisation parameter T: 4
    Optimisation Particle mask diameter: 320
    Optimisation Mask individual particles with zeros: No
    Optimisation Reference mask:
    Sampling Angular sampling interval: 7.5
    Sampling Offset search range: 6
    Sampling Offset search step: 2
    Sampling Perform angular searches: No
    Sampling Local angular search range:
    Running Number of MPI procs: 14
    Running Number of threads: 8
    Running Submit to queue: Yes
    Running Queue name: /usr8/repository/relion/relion-1.4-gyan/bin/qsub.csh
    Running Queue Submit Command: qsub
    Running Standard submission script: /usr8/repository/relion/relion-1.4/bin/qsub.csh
    Running Additional arguments:

    Run Relion GUI will submit the following command: Executing: csh run1/relion_submit.script &

    which relion_refine_mpi`
    --o    run1/relion
    --i    /usr8/spider/test/d_relion3/input/relion_1_data.star
    --particle_diameter    320
    --angpix    2.78
    --ref    /usr8/spider/test/d_relion3/input/REF_Control_80S_relion.fmr
    --firstiter_cc
    --ini_high    60
    --iter    25
    --tau2_fudge    4
    --flatten_solvent
    --zero_mask
    --ctf
    --sym    C1
    --K    4
    --oversampling    1
    --healpix_order    2
    --offset_range    6
    --offset_step    4
    --norm   
    --scale   
    --j    8
  5. Convert output files from 'MRC' to SPIDER format.

    Use: from_mrc.spi to read:
    relion_it###_half+_class***MRC class average file
    and create:
    ¤ spi_it###_half+_class***

    Example:

    ¤ spider spi/mrc @from_mrc    Create SPIDER format files

Source: spi2relion.html     Page updated: 04/25/16     ArDean Leith

Enquiries: spider@wadsworth.org