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.

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

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.

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 --minvol=-1 --maxvol=-1 --delta=30.0 --sym=c2 --rmedge --rmgold --boxsz=-1 --threads=12