EMAN2 Tomography Workflow Tutorial

Computer Requirements

Download Data

Prepare input files (~2 minutes)

Project Manager

For your own data:

Tiltseries Alignment and Tomogram Reconstruction (20 min)

Alignment of the tilt-series is performed iteratively in conjunction with tomogram reconstruction. Tomograms are not normally reconstructed at full resolution, generally limited to 1k x 1k or 2k x 2k, but the tilt-series are aligned at full resolution. For high resolution subtomogram averaging, the raw tilt-series data is used, based on coordinates from particle picking in the downsampled tomograms. On a typical workstation reconstruction takes about 4-5 minutes per tomogram.

For the tutorial tilt-series:

Tomogram reconstruction

For your own data:

CTF Estimation (10 min)

For the tutorial tilt-series:

When working with your own data:

Note that this program is only estimating CTF parameters, taking tilt into account. It is not performing any phase-flipping corrections on whole tomograms. CTF correction is performed later as a per-particle process. This process requires metadata determined during tilt-series alignment, so it cannot be used with tomograms reconstructed using other software packages.

Tomogram evaluation (optional)

Tomogram evaluation

Analysis and visualization -> Evaluate tomograms can be used to evaluate the quality of your tilt series alignments and tomogram reconstructions. This tool will show more information as you progress through the tutorial, but can be used already at this point to make various assessments of your tomograms.

Tomogram annotation (optional)

2D particle picking

This section is brief and is only an update to the more detailed tutorial: TomoSeg. Some directory structure and user interfaces have changed in the latest version to match new tomogram workflow as described here:

Particle picking (10-15 min)

3D particle picking

Particle extraction (a few min)

In this pipeline, the full 1k or 2k tomograms are used only as a reference to identify the location of the objects to be averaged. Now that we have particle locations, the software returns to the original tilt-series, extracts a per-particle tilt-series, and reconstructs each particle in 3-D independently.

For the tutorial tilt-series:

For your own data:

Initial model generation (10 - 60 min)

Initial model generation

While intuitively it seems like, since the particles are already in 3-D, that the concept of an "initial model" should not be necessary. Unfortunately, due to the missing wedge, and the low resolution of one individual particle (particularly from cells), it is actually critical to make a good starting average, and historically it has been challenging to get a good one, depending on the shape of the molecule. This new procedure based on stochastic gradient descent has proven to be quite robust, but it is difficult for the computer to tell when it has converged sufficiently. For this reason, the default behavior is to run much longer than is normally required, and have a human decide when it's "good enough" and terminate the process. If you use a small shrink value and let it run to completion, it can take some time to run, but this is normally a waste.

For the tutorial tilt-series:

For your own data:

Template matching (5 min)

In this step, we will use the initial model you just produced as a template for finding all of the ribosomes in all 4 tomograms. If you completed the Tomogram Annotation step above, and have already extracted a full set of 1000+ particles, then you can skip this step, as we already have all of the particles. Note that here, and everywhere else in the tomography pipeline, reconstructed particles have positive contrast (look white in projection) and tomograms/tilt series have negative contrast (look dark in projection). If you wish to use a reference volume from the PDB or somesuch, then it should have positive contrast as is normal in the single particle CryoEM field.

Particle extraction (~1 hour)

Again, if you already did Tomogram Annotation above, this step isn't necessary. It is only required if you just did Template Matching.

Since this involves several thousand particles instead of 30-50, it will take quite a lot longer to run. The actual time will depend partially on the speed of your storage.

For the tutorial tilt-series:

Subtomogram refinement (~6 hr)

3D refinement

This step performs a conventional iterative subtomogram averaging using the full set of particles. Typically it will achieve resolutions in the 15-25 A range with a reasonable number of particles. As it involves 3-D alignment of the full set of particles multiple times, it takes a significant amount of compute time. Higher resolutions are achieved in the next stage after this (subtilt refinement).

For the tutorial tilt-series:

Results will gradually appear in spt_XX/

For your own data:

Subtilt refinement (~32 hr)

Subtilt refinement directory

With the results of a good subtomogram alignment/average, we are now ready to switch to alignment of the individual particle images in each tilt, along with per-particle-per-tilt CTF correction and other refinements. This is effectively a hybrid of single particle analysis and subtomogram averaging, and can readily achieve subnanometer resolution IF the data is of sufficient quality. The tutorial data set is, but many cellular tomograms, for example, are not collected with high resolution in mind, and even with this sort of refinement will be unable to achieve resolutions better than 10-30 A, depending on the data. This process is completely automatic, based on all of the metadata collected up to this point. While it is possible to perform "subtomogram refinement" with subtomograms from any tomogram, Subtilt Refinement cannot operate properly unless all preceding steps occurred within EMAN2.

For the tutorial tilt series:

For your own data:

Congratulations! The final result of the tutorial will be found in "subtlt_00/". The final 3-D map will be "threed_04.hdf" with the default parameters. The final gold standard resolution curve will be "fsc_maskedtight_04.txt". The optional steps below are tools you can use to evaluate your results in more detail.

Refinement evaluation (optional)

Refinement evaluation This tool helps visualize and compare results from multiple subtomogram refinement runs.

EMAN2/e2tomo (last edited 2022-08-29 14:20:27 by SteveLudtke)