This tutorial uses a public in situ CryoET dataset ( EMPIAR-11830) of Chlamydomonas reinhardtii prepared using cryo-plasmaFIB milling. Here, we use 5 tilt series and target the structure and dynamics of ATP synthase inside mitochondria.

It is recommended to cross reference with previous tutorials of ribosomes and viruses .

Many of the functions here require an EMAN2 installation after 06/2026. A newer version is almost always better. A latest continuous build may work but compiling from source is preferred. For the installation guide, see here.

Make a new empty folder for the project and 'cd' into that folder. Make sure all EMAN2 commands you run are executed from within this folder.

Unzip the dataset, and you should have a folder called “tiltseries”, with four hdf image stacks in it, inside the project folder. To view the tilt series, run e2display.py, locate the file in the browser, and click Show2D.

Start from one good tilt series to play with the parameters for tilt series alignment and tomogram reconstruction. After having a good set of options, we will run the program with the same parameters for all tilt series later.

e2tomogram.py tiltseries/06092023_NNPK_Arctis_WebUI_Ron_data_Position_4.hdf --tltstep=3.0 --npk=10 --tltkeep=0.9 --outsize=1k --niter=2 --bytile --pkkeep=0.9 --compressbits=8 --clipz=320 --bxsz=32 --filterres=50 --threads=12 --patchtrack=2

Before reconstructing all tilt series, we need to make sure the handedness of the tomogram is correct.

e2spt_tomoctf.py tiltseries/06092023_NNPK_Arctis_WebUI_Ron_data_Position_4.hdf --dfrange=2.0,7.0,0.02 --psrange=10,15,5 --tilesize=400 --voltage=300 --cs=2.7 --nref=15 --stepx=20 --stepy=40 --checkhand --threads=1 --writetmp

The handedness of the tilt series should be correct.

Reconstruct all tilt series using the same parameters and the tilt axis estimated by the handedness check. Note that in the full dataset, tilt series of different sessions in EMPIAR-11830 may have different tilt step and pixel size in their header. Run the –alltiltseries command with caution when processing large datasets. The 5 tilt series in this tutorial are from the same session and have similar conditions. While the selection makes the processing simpler, it limits the final resolution because the 5 tilt series are collected at similar (and relatively high) defocus.

e2tomogram.py --alltiltseries --tltax -83.5 --tltstep=3.0 --npk=10 --tltkeep=0.9 --outsize=1k --niter=2 --bytile --pkkeep=0.9 --compressbits=8 --clipz=320 --bxsz=32 --filterres=50 --threads=12 --patchtrack=2 --notmp

Now we estimate the defocus for all tilt series. Note here we only estimate the CTF, not correct for it. The correction will happen later when extracting particles. To get to high resolution, it is often safer to use a higher than the default `–tilesize`.

e2spt_tomoctf.py --alltiltseries --dfrange=2.0,7.0,0.02 --psrange=10,15,5 --tilesize=400 --voltage=300 --cs=2.7 --nref=15 --stepx=20 --stepy=40 --threads=5

First, evaluate the tomograms using

e2tomo_eval.py

Here we pick a few particles manually. In this dataset, we just need ~70 particles to make a good initial model. Here we label them as atpsyn_init.

Extract the manually selected particles and build set first.

e2spt_extract.py --boxsz_unbin=288 --label=atpsyn_init --threads=8 --alltomograms --shrink=4 --tltkeep=1.0 --compressbits=8 --parallel thread:5
e2spt_buildsets.py --allparticles

Now make the initial model.

e2spt_sgd_new.py sets/atpsyn_init_bin4.lst --res=60.0 --niter=50 --shrink=1 --parallel=thread:64 --ncls=3 --batch=12 --learnrate=0.2 --sym=c1

Note the structure should be c2 symmetrical. At this point, it is recommended to rotate the initial model to the symmetry axis to take advantage of the symmetry in later steps. Sometimes, this can be done automatically.

e2symsearch.py sptsgd_00/output_cls0.hdf --sym c2 --applysym

However, this program is designed for higher resolution structure. Since the resolution is low and the symmetry in situ structure is not perfect, this may fail. One solution is to rotate the volume roughly to the symmtry axis in FilterTool, using the xform processor. Here I launched the FilterTool in safe mode (hold SHIFT), and used “xform:alt=30:az=50”. The checkboxs afront unused parameters need to be unchecked. Save the processed map and rename it to sptsgd_00/output_cls0_xf.hdf. Then run e2symsearch.py in refine mode to optimize the symmetry axis.

e2symsearch.py sptsgd_00/output_cls0_xf.hdf --sym c2 --applysym --refineonly

Alternatively, it is also possible to skip this step for now and perform the initial refinement with c1. Automatic symmetry search is more likely to converge once the structure reaches higher resolution.

Pick particles using the initial model generated in the last step.

e2spt_tempmatch.py tomograms/*.hdf --reference=sptsgd_00/output_cls0_xf_sym.hdf --label=atpsyn --nptcl=1500 --dthr=60.0 --vthr=6.0 --delta=30.0 --sym=c2 --rmedge --rmgold --threads=12

The template matching should work fine inside mitochondria but leave false positives outside. Since we only have 5 tomograms here, it is easy to take a look and manually clean the particles. Lauch the manual boxer and use the Eraser tool to remove particles outside the mitochondria. Uncheck “Limit Side Boxes” will show all boxes along one axis in each view so particles on the edge across all depth can be removed with one click.

After the clean up, I got ~5000 particles total.

For the full dataset, using the deep learning based particle picker can reduce the manual effort of the cleaning step. Because the ATP synthase has quite distinct top vs side view, training the model can take multiple iterations of training set refinement and not efficient for a small dataset. Please refer to previous tutorials for details.

The recently made contrast base tilt series alignment refinement tool can often improve the resolution of averaged structures. This step can be done right after the initial tilt series alignment step, but when particle coordinate information exist in the tomogram, the program will specifically enhance the contrast in region with particles. This might lead to slightly better performance.

e2tomogram_refine.py tiltseries/*.hdf

First extract the particles from template matching. With this tutorial dataset, we expect to get ~10Å resolution, so shrink by 2 should be sufficient.

e2spt_extract.py --boxsz_unbin=256 --label=atpsyn --threads=12 --maxtilt=100 --padtwod=2.0 --alltomograms --shrink=2.0 --tltkeep=1.0 --compressbits=8 --parallel thread:5

Now run build set and start the initial round of refinement. Note here we use the 'g' iteration introduced in 2026. It is much faster than 't' and produces higher resolution final structure in most cases. My current recommendation is alternating 'p' and 'g' iterations but ends with a 'p'. The 'g' iterations may have a slightly lower FSC curve itself sometimes but still improve the FSC of the next 'p' iteration.

e2spt_refine_new.py --ptcls=sets/atpsyn_bin2.lst --ref=sptsgd_00/output_cls0_xf_sym.hdf --startres=50.0 --goldstandard --sym=c2 --iters=p2,g,p,g,p --keep=0.95 --parallel=thread:64 --threads=64

The first refinement should bring the resolution to around 15Å. If the structure is not aligned to symmetry axis yet, this is a good time to align it. Then we can set up the next refinement run, with the same particles and reference, but with a specified mask and local resolution based filter. The mask can be made in filtertool manually to keep only one pair of ATP synthase for alignment. I used mask.zeroedge3d followed by mask.auto3d.thresh to add some padding and a soft falloff.

e2spt_refine_new.py --ptcls sets/atpsyn_bin2.lst --ref=spt_00/threed_07.hdf --startres=25 --goldstandard --sym=c2 --iters=p2,g,p,g,p --keep=0.95 --parallel=thread:96 --threads=96 --mask mask_00.hdf --tophat localwiener

This should bring the resolution to about 12Å.

Since we want to look at dynamics of ATP synthase, we need to first determine the structure as monomers. To do this, first expand the symmetry of the particles, making 2 copies of each particle at the c2 positions. For no good reason, some options of e2proclst.py work in place and some work on new lists created.

cp spt_01/aliptcls3d_06.lst spt_01/aliptcls3d_06_sym.lst
e2proclst.py spt_01/aliptcls3d_06_sym.lst --sym c2

The new list, spt_01/aliptcls3d_06_sym.lst should have 2x particles. While not necessary, it is better to shift on of the two asymmetrical units to the center of the box, and roughly align it so the central stalk is along the z axis. This can be done in the FilterTool with the xform processor, and let's call the output spt_01/threed_06_xf.hdf. To also modify the alignment of the particles, first get the exact alignment in between, then apply it to the particle list.

e2proc3d.py spt_01/threed_06.hdf spt_01/threed_06_ali.hdf --align rotate_translate_3d_tree --alignref spt_01/threed_06_xf.hdf
e2proclst.py spt_01/aliptcls3d_06_sym.lst --create spt_01/aliptcls3d_06_sym_xf.lst --applyxf spt_01/threed_06_ali.hdf

To make sure the operations on particle lists are done properly, compare the Euler angles of the lists by clicking “Plot2D”.

Finally we can refine the monomer particles. Here we also need to make a customized mask for the monomer, keeping only one ATP synthase inside the mask. This is also done in FilterTool using mask.zeroedge3d followed by mask.auto3d.thresh. Call this mask mask_01.hdf.

e2spt_gathermeta.py --ptcls spt_01/aliptcls3d_06_sym_xf.lst --ali2d spt_01/aliptcls2d_06.lst
e2spt_refine_new.py --path spt_02 --continuefrom 0.5 --localrefine --mask mask_01.hdf --setsf sf.txt --iters=p2,g,p,g,p --keep=0.95 --parallel=thread:64 --tophat localwiener

The resolution should reach 11Å by the end of this.

Before heterogeneity analysis, we need to get rid of the bad particles first. While they are automatically downweighted and having a small fraction of bad particles generally does not have a strong impact in the refinement, the heterogeneity analysis might interpret the good vs bad particles as actual structural differences. Although we have manually removed the obvious bad particles outside mitochondria earlier, there are still some that are just bare cristae membranes. Here we remove them through simple classification.

e2spt_sgd_new.py spt_02/aliptcls3d_07.lst --res=50.0 --niter=100 --shrink=1 --parallel=thread:64 --ncls=2 --batch=12 --learnrate=0.2 --sym=c1 --classify --refine --skipali

This generates one correct structure of ATP synthase, and the other looks somewhat flat, likely a piece of misaligned membrane. However, e2spt_sgd_new.py does not actually assign particles to classes, so we need to run a full classification with all particles.

e2spt_refinemulti_new.py sptsgd_01/output_cls0.hdf sptsgd_01/output_cls1.hdf --ptcls spt_02/aliptcls3d_07.lst --niter 3 --maxres 20 --loadali3d --skipali --loadali2d spt_02/aliptcls2d_07.lst --parallel thread:64

e2spt_gathermeta.py --ptcls sptcls_00/aliptcls3d_02_00.lst --ali2d spt_02/aliptcls2d_06.lst
e2spt_refine_new.py --path spt_03 --continuefrom 0.5 --localrefine --mask mask_01.hdf --setsf sf.txt --iters=p2,g,p,g,p --keep=0.95 --parallel=thread:64 --tophat localwiener

First convert the voxel map into GMM representation, and start a global refinement.

e2gmm_guess_n.py spt_03/threed_07.hdf --thr 4 --maxres 11 --evenodd --startn 4000 --jax
e2gmm_spt_refine_iter.py spt_02/threed_07.hdf --initpts spt_02/threed_07_seg.pdb --startres 11 --maskpp mask_01.hdf

The resolution should get slightly better than 10Å at this point. Next, we can focus the refinement on the rotation of F1 head. Make a mask using FilterTool that covers the F1 head only, and name it mask_f1.hdf. Here we first run one iteration of the deep learning based alignment to recover the large scale rotation, followed by 3 iterations of the direct alignment from the deep learning result.

e2gmm_spt_refine_iter.py gmm_00/threed_03.hdf --initpts spt_03/threed_07_seg.pdb --startres 15 --maskpp mask_01.hdf --mask mask_f1.hdf --align_mlp --niter 1
e2gmm_spt_refine_iter.py gmm_01/threed_01.hdf --initpts spt_03/threed_07_seg.pdb --startres 10 --maskpp mask_01.hdf --mask mask_f1.hdf

This should improve the structure features at the F1 head domain, but the FSC resolution does not necessarily improve here. Because the even/odd half set only are only aligned to the “neutral” struture of their half-set and never see each other, there is a possiblity that they converge to slightly different states, and the FSC resolution decrease even though the feature in each half-set improves. This is less of a problem in datasets with more particles since the “neutral” state would be better defined, but here there are some uncertainties with only 5 tomograms…

To visualize the dynamics, run the following.

e2gmm_eval.py --pts gmm_01/mid_01_even.txt --pcaout gmm_01/pca_even.txt --ncls 4 --spt --ptclsin gmm_01/aliptcls2d_00_even.lst --ptclsout gmm_01/class_01_even.lst --mode regress --outsize 128 --parallel thread:64 --nptcl 800
e2proc3d.py gmm_01/class_01_even.hdf gmm_01/class_01_even_lp.hdf --process filter.lowpass.gauss:cutoff_freq=0.067 --process normalize

This only shows the motion of the even set, and the same can be done to the odd half. Since the deep learning models for the two half-sets are trained independently, visualizing the motion in the combined dataset without breaking the “gold-standard” validation is impossible. Still, the rotation movement should be visible already even with the small dataset.