FAQs

General

How do you use RELION?

Read our Recommended_procedures! We used these procedures for our DNA origami structure, as well as for our near-atomic ribosome structures.

Preprocessing

Do I need to downsize my images?

RELION will estimate the resolution of your model and downsize the images internally. Therefore, using downsized images will NOT be (much) faster than using large images. In general it is therefore NOT recommended to use downsized images: it could limit your resolution. The only reason why one would want to downsize images if they are too big (e.g. 800x800 pixels) for the reconstruction(s) to fit into memory. If this is the case, you can use the Preprocessing procedure to downsize your images.

Classification

Do you have an example of how to run 3D classification?

Yes, see the Classification example.

Should I refine my 3D classes in a different program to reach higher resolution?

This is not necessary: you may do so in RELION. Higher resolution often means using fine orientational and translational samplings, which may be prohibitive (speed and memory-wise) inside classification runs. One can however write out a new STAR file that contains only the images belonging to a certain class (see the next FAQ). That STAR file could then be used for a single-reference refinement with fine samplings in RELION.

How can I select images from a STAR file?

The following awk command may be used to select images belonging to classes number 3 or 4 (assuming rlnClassNumber is the 13th column):

awk '{if (NF<= 2) {print} else {if ($13==3 || $13==4) print }}' < classify_it000025_data.star > class3_only_input.star

Some knowledge of awk is REALLY useful! This light awk introduction may be helpful. The command above prints all lines if the number of fields (NF) is 1 (i.e. for the header), and else (i.e. for the data block) it will only print those lines if the 13th column ($13) equals 3 or 4. Note one may change the "==" sign for ">", "<=", etc. That way, one could select images above a certain MaxValueProbDistribution, within a certain AnglePsi range, etc.

3D refinement

How can I make a plot of the orientational distribution of my particles?

RELION outputs all the information in the STAR files for each iteration, but at the moment will not make a plot of this. You can use the make_orientational_distribution_plot.csh script to make a BILD file (using XMIPP), which can then be read into UCSF Chimera.

I have run RELION but get a non-sense map as a result, e.g. a spherical blob

Make sure the following things are correct:

• You have normalized all particles to zero-mean background with a standard deviation in the noise of one.
• Your STAR file header is correct: each label corresponds to the correct column
• You have used the correct pixel size
• There are no particles with very large or very small pixel values.
• You have indicated the starting map is not on the correct greyscale (if the map does not come from RELION itself or XMIPP)

(The first three items on this list are all taken care of if you use the PreProcessing GUI

Upon restarting I get an error: incorrect table model_group_xx

Make sure all groups have at least 20-50 particles in them. The error above is likely to an empty group for one of the two independent halves of the data. Note you can join multiple micrographs into one group by giving them the same rlnMicrographName in the input STAR file. If you do this, try to join micrographs with similar defocus values and similar apparent signal-to-noise ratios.

The resolution of the RELION output map is lower than I expected

There are two answers to this. Firstly, if your expectations are based on refinement in a different program, and that program does not strictly prevent overfitting, then it might be that your expectations are wrong: perhaps the other program overfitted your data and therefore has given you a false high-resolution estimate. Secondly, we have now observed that RELION slightly underestimates resolution. However, you may still get your high-resolution map as explained on the Analyse results page. Please write to us if you genuinely believe RELION has done a bad job at refining your structure. Perhaps we may learn how to improve RELION from your case.

Computational issues

My runs keep crashing at seemingly random points in the refinement

We noticed similar problems on our Dell cluster. Things became much more stable when we switched off TCP segmentation offloading, by using the command mentioned on this linux page.

Please note that due to limited resources we cannot provide support related to high-performance computing issues... If your jobs seem to die at seemingly random points, please do not email us, but speak to your system administrator... RELION makes use of some pretty intensive high-performance computing, and setting this up satisfactorily is not always straightforward.

How can I minimise computational requirements?

See this page for an explanation about the computational requirements. Understanding these may help you make more informed decisions on how to minimise running costs. More information is also given in the 2012 JSB paper.