Working with Cryo-EM Software

This guide compiles practical tips and insights we've accumulated from working with cryo-EM software during our research. It covers essential software tools commonly used in our work, including ChimeraX, RELION, EMAN2, and CryoSPARC.

ChimeraX

ChimeraX provides many useful commands for reference. For a comprehensive collection of recipes and examples, visit ChimeraX Recipes.

Flipping a Volume

To mirror-flip a volume in ChimeraX:

vol flip #1

Displaying Coordinate Axes

To visualize coordinate axes, create an XYZ-axes.bild file with the following content and open it in ChimeraX:

.comment -- This file shows X,Y,Z axes as red, yellow, blue arrows --
.comment -- Edit "translate" and "scale" values to adjust offset and size --
.translate 0.0 0.0 0.0
.scale 5
.sphere 0 0 0 0.5
.color 1 0 0
.arrow 0 0 0 96 0 0 0.7
.color 1 1 0
.arrow 0 0 0 0 96 0 0.7
.color 0 0 1
.arrow 0 0 0 0 0 96 0.7

Viewing Multiple Models or Maps

Viewing atomic models: Use the mseries command. See the official documentation for details.
Viewing density maps (volumes): Use the vseries command. See the official documentation for details.

EMAN2

Calculating and Plotting FSC Curves

EMAN2 provides tools for calculating FSC (Fourier Shell Correlation) curves:

# Activate the EMAN2 conda environment
e2proc3d.py a.mrc output.txt --calcfsc=b.mrc

To visualize the results, use the separate plotfsc.py script. The official example script is available at plotfsc.py. Run:

python plotfsc.py output.txt

RELION

Low-pass Filtering a Volume

To apply a low-pass filter to a volume:

relion_image_handler --angpix xxx --lowpass 20 --i input.mrc --o output.mrc

Down-sampling (Binning) a Volume

To down-sample (bin) a volume:

relion_image_handler --angpix xxx --rescale_angpix xxx --new_box xxx --i input.mrc --o output.mrc

Simulating the Projection Process

To simulate projections, you need a STAR file containing pose information:

relion_project --i input.mrc --o rln_sim --ctf --angpix xxx --ang example.star

# Add noise (0.1 is high enough for most cases)
relion_project --i input.mrc --o rln_sim --ctf --angpix xxx --add_noise --white_noise 0.01 --ang test.star

Reconstruction

To perform reconstruction from particle STAR files:

relion_reconstruct --i test.star --o relion.mrc --ctf --sym C1 --angpix xxx --mask_diameter xxx --fsc

# Using MPI for parallel processing
mpirun -n 20 `which relion_reconstruct_mpi` --i test.star --o relion.mrc --ctf --sym C1 --angpix xxx --mask_diameter xxx --fsc