Classification example

10,000 70S ribosomes wi/wo EFG

The test data set that will be used below was provided by Joachim Frank and is stored at the EBI-EMDB.

Download data and reference

Download the test data set from here, as well as the corresponding metadata that is stored in this PDF file. Save both files in your working directory.

EMDB entry 1056 will be used as initial reference. Save this file in the same working directory. Note this reference has the same pixel size (2.8A) and the same box size (130x130) as the data set above. Therefore, no re-scaling or windowing operations are necessary. Although this map is at a resolution of ~9 A, note that it will be low-pass filtered to 80 A in the RELION run below.

Unpack the data as follows:

tar -xf J-Frank_70s_real_data.tar
gunzip emd_1056.map.gz
mv emd_1056.map emd_1056.mrc

Normalise the images

The data come at an arbitrary density scale. Make sure the background density is on average approximately zero by normalising the data. The following XMIPP commands (version 2.4) set the average density of each image to 0 and the standard deviation to 1.

xmipp_selfile_create "win/*dat" >all.sel
xmipp_normalize -i all.sel -method OldXmipp 

Note that any other package may be used to do a similar thing, the most important thing is that the average background density should be approximately ZERO.

Prepare the input STAR file

From the PDF provided by Joachim Frank select the following (14) lines and save them in a text file called defocus.dat

1 3 1347.0 1347.0 21580.
2 3 505.00 1852.0 24833.
3 3 989.00 2841.0 26450.
4 3 857.00 3698.0 28320.
5 3 475.00 4173.0 30993.
6 3 349.00 4522.0 33150.
7 3 478.00 5000.0 34588.
8 3 1242.0 6242.0 21580.
9 3 713.00 6955.0 24833.
10 3 1255.0 8210.0 26450.
11 3 1022.0 9232.0 28320.
12 3 304.00 9536.0 30993.
13 3 232.00 9768.0 33150.
14 3 232.00 10000. 34588.

Then save the following lines as a file called make_star.csh

#!/usr/bin/env csh
ls -l win/*dat | awk '{print $NF}' >imagelist
#
relion_star_loopheader rlnImageName rlnMicrographName rlnDefocusU rlnVoltage rlnSphericalAberration rlnAmplitudeContrast > all_images.star
#
set ngr = 14
set gr = 0
while ($gr < $ngr)
 @ gr++
 set nn=`head -n $gr defocus.dat  | tail -1 | awk '{print int($3)}'`
 set tot=`head -n $gr defocus.dat | tail -1 | awk '{print int($4)}'`
 set def=`head -n $gr defocus.dat | tail -1 | awk '{print $5}'`
 head -n ${tot} imagelist | tail -n ${nn} |awk -v"def=$def" -v"gr=$gr" '{print $1, gr, def, 200, 2, 0.1}' >> all_images.star
end

And execute it to generate the input STAR file with all image names and CTF information, using the command:

csh make_star.csh

Run RELION

Save the following lines in a file called example_gui3d.settings:

is_continue == false
Output rootname: == example/K4
Continue from here:  == 
Input images: == /lmb/home/scheres/work/relion/ribo_test_case/all_images.star
Reference map: == /lmb/home/scheres/work/relion/ribo_test_case/emd_1056.mrc
Padding factor: == 3
Ref. map is on absolute greyscale? == No
Initial low-pass filter (A): == 80
Particle diameter (A): == 340
Pixel size (A): == 2.8
Number of iterations: == 25
Regularisation parameter: == 4
Do solvent flattening? == Yes
Solvent mask: == 
Do normalisation correction? == Yes
Do intensity correction? == Yes
Do CTF-correction? == Yes
Only flip phases? == No
Have data been phase-flipped? == No
Ignore CTFs until first peak? == No
Has reference been CTF-corrected? == No
Symmetry group: == C
Symmetry number: == 1
Angular sampling interval (deg): == 7.5 degrees
Limit the tilt angle? == No
Limited tilt angle: == -91
Perform local angular searches? == No
Local angular search range: == 5
Number of classes: == 4
Oversampling order: == 1
Offset search range (pix): == 10
Offset search step (pix): == 1
Number of MPI procs: == 8
Number of threads: == 8
Submit to queue? == Yes
Queue name:  == openmpi_8
Queue submit command: == qsub
Standard submission script: == /lmb/home/scheres/app/relion/gui/qsub.csh
Additional arguments: ==  --random_seed 1

Start the RELION GUI by typing relion from the command line (inside the working directory). Load the example_gui3d.settings through the File Menu option. Then in the Running tab adapt the parameters for your particular cluster setup, and submit the job by clicking on the orange "Run!" button.

That's it! Just wait until your job is finished. Using 8 MPI nodes, each with 8 threads, this calculation took XXX wall-clock hours on the LMB cluster.