• Most programs are available in EMAN2 builds after 2026-06, but newer versions are typically better.
• The tutorial is only tested on Linux with Nvidia GPU and CUDA.
• For reference of the method, see the preprint.
• A separate tutorial about the same method applied to CryoET data can be found here.

In this tutorial, we use a refinement of TRPV1 from a CryoSPARC tutorial. In theory you can also do everything in EMAN2 from scratch, but this makes a clearer comparison with other software.

Start from the consensus refinement from CryoSPARC, and import the particles with:

e2convertrelion.py J35_008_particles.star --onestack particles/all_particles.hdf

Depending on the masking, the resolution should be around 2.7 to 3 Å. After the import finishes, you should find relavent files in the r3d_00 folder.

First build the GMM representation from the 3D volume.

e2gmm_guess_n.py r3d_00/threed_00.hdf --evenodd --nopdb --thr 4.5 --maxres 3

Here the –evenodd option will build GMM for the two half-set separately. Pick a threshold for –thr so all protein density of interest are visible. –nopdb force it to write points as txt instead of pdb, since the pdb format limits the maximum number of atoms. This should produce r3d_00/threed_00_gmm_even/odd.txt.

Run the global refinement next.

e2gmm_refine_iter.py r3d_00/threed_00.hdf --startres 2.8 --initpts r3d_00/threed_00_gmm.txt --maskpp r3d_00/mask.hdf --jax --niter 3 --parallel thread:32 --sym c4

It is ok to use r3d_00/threed_00_gmm.txt even though the file does not exist, since the program will look for the even/odd versions automatically. Make sure to add –jax to call the JAX backend, as we are retiring the Tensorflow backend gradually. Without –maskpp, the program will use auto-generated masks. Here we keep the same masking for better comparison.

After the refinement, the global resolution should reach Nyquist, at 2.5 Å, according to the “gold-standard” FSC.

Now we run focused refinement on the CTD of one asymmetrical unit. First expand the c4 symmetry. Note that e2proclst.py –sym operates in place, so it is safer to backup.

e2proclst.py gmm_00/ptcls_03_even.lst --sym c4
e2proclst.py gmm_00/ptcls_03_odd.lst --sym c4