Single Particle Tomography in EMAN2

Wednesday March 16, 2011 - P.M. Practical EMAN2 SPT tutorial

SPT (single particle tomography) capabilities are relatively new in EMAN2. They were inspired by Michael Schmid's studies on sub-volume averaging (mostly on viruses), and a stubborn student's insistence on doing extensive sub-volume averaging on chaperons.

This session will cover the beta version of a small fraction of the possibilities for SPT processing EMAN2 will eventually offer.

OUTLINE

• 1) SPT processing through EMAN2's workflow (e2workflow.py)
• 2) Sub-volume extraction from tomograms using EMAN2's 3D particle picking tool (e2tomoboxer.py)

• 3) "Preparation" of extracted particles for alignment. [Details later. For a myriad of reasons, it is NOT recommendable to align and average sub-volumes directly after extraction without "preparing" them first].

• 4) Reference-based alignment and averaging.

  PLEASE NOTE that "particle" and "sub-volume" are used interchangeably

SOFTWARE

All necessary software is provided as part of EMAN2. If you don't have EMAN2 installed, you can download the most updated version (for your specific platform, Windows, Linux or Mac), from here:

http://ncmi.bcm.edu/ncmi/software/counter_222/software_86

RAW DATA

We have prepared sample data for this tutorial. The tomogram in the link below comes from a tilt series of epsilon15 virus particles in vitro, recorded using Zernike phase-plate technology:

e15phaseplate.rec

The following tomogram also comes from a tilt series of epsilon15 viruses in vitro but was recorded under conventional cryoEM imaging conditions:

e15normal.rec

BOXING

There are two options for opening the tomogram for purposes of boxing it.

1) Directly, by typing e2tomoboxer.py followed my the path to the tomogram file at the commandline.

2) Or you can launch e2workflow.py from the commandline and access a tomogram through the browser in the tomographic menu.

[In theory, you can open a tomogram for contemplation purposes by typing: "e2display.py <my_tomogram_path_name_goes_here>" at the commandline. This is NOT recommendable, unless you have a grossly large (VERY, VERY large) amount of virtual memory on your computer; otherwise, catastrophe WILL befall upon you].

Let's explore the FIRST APPROACH.

OPENING A TOMOGRAM DIRECTLY WITH e2tomoboxer.py

To launch one of the tomograms provided (you're free to choose which ever you prefer) type the following at the commandline:

e2tomoboxer.py e15normal.rec --yshort --inmemory

To specify --yshort, or not to

• IF your tomogram has its smallest/slimmest dimension running along the Y-axis, this option will FLIP the tomogram such that the smallest dimension becomes parallel to the Z-axis.

What does this mean?

• Some tomograms are built with the slimmest part of the 3D volume (that corresponding to the "ice thickness" in cryoEM) running along the Y-axis. By convention, EMAN2 displays things such that the Z-axis is perpendicular to the screen. Ideally, you would want the slimmest part of the volume to be aligned along Z so you can view the entire tomogram from the "TOP" and look at the entire area in the XY plane slice by slice, as you go through the volume.

USING THE WORKFLOW FOR SPT