Single Particle Tomography in EMAN2

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

There is no Wiki version of the tutorial as of now. You MUST download it as a PDF or word document, provided below.

The links SHOULD work directly (it is safe to do right-click followed by "save as"). If not, once you click on the link, it will take you to another page where you'll see a "DOWNLOAD" link. Click on that to get the corresponding file.

TUTORIAL DOCUMENT

eman2_spt_tutorial.pdf

eman2_spt_tutorial.docx

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

PREPARED COORDINATES FILES

e15phaseplate_coords.txt

e15normal_coords.txt

REFERENCES

e15ref_prep_icos_thr_norm.hdf

e15ref_prep_asym_thr_norm.hdf

e15reference_raw.hdf

ANYTHING BELOW THIS MARK IS DEPRECATED

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

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 the GUI that will show you the tomogram (you're free to choose whichever you prefer, "e15normal.rec" or "e15phaseplate.rec") type the following COMMAND #001 at the commandline:

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

• EXPLANATION

  To specify --yshort, or not to

  • This option will FLIP the orientation of your tomogram respect to the Y and Z coordinates. You should ONLY specify it IF the tomogram has its smallest/slimmest dimension running along the Y-axis, so that it becomes parallel to the Z-axis instead.

  Ok... but WHY? 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. BUT in EMAN2 volumes are displayed such that the Z-axis is perpendicular to the screen. If you open one of these tomograms "as is", you'll be looking at it "from the side". Most of the time you want the slimmest part of the volume to be aligned along Z (NOT Y), so you can see the tomogram "from the TOP", and look at the entire CCD-captured area in the XY plane slice by slice, as you go through the volume.

  --inmemory

  • This option pre-loads your tomogram to memory, allowing to box particles (or do whatever you want to do in e2tomoboxer) more smoothly (faster), because reading data from memory is faster than reading it from disk. You can get the entire list of options that e2tomoboxer.py takes (not many at this point) and some sort of explanation of what they're for by typing the following at the commandline:

      e2tomoboxer.py -h

If you ran the command and took a leap of faith in skipping the explanation, you will probably have already clicked a few things in the GUI (Graphical User Interface with nice 'clickable' buttons) that popped up. (I hope you did; extensive explanations really ought to be read only when following the execution boxes doesn't work smoothly, when you're confused, when you have a lot of time to spare and nothing better to do, or when you're a purist [slightly OCD] about reading manuals in their entirety with the noble purpose of getting the most out of them).

USING THE WORKFLOW FOR SPT

The e2tomoboxer.py GUI should look like this:

This window is called

You will notice...