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