Human T-cell Chimera Demonstration Notes

Tom Goddard
December 13, 2006.
Presented at Workshop for Visualization of Cellular Complexity
Max Planck Institute, Martinsried, Germany.

This demonstration shows electron microscope tomography of a human immune system cell in contact with target cell. Vesicles containing serine proteases are transported to the cell membrane along microtubles to kill the target cell. The demonstration shows the tomography map, a segmentation of the tomography, a high resolution microtubule map, and a microtubule atomic model. The demonstration illustrates use of several Chimera tools: map display, single plane display, placing markers on maps, segmentation display, tracing paths, fitting maps in other maps, fitting models in maps, multimeric atomic model display, display of inter-molecular atomic contacts.


Step by step notes for demonstrating visualization form cellular scale to atomic scale.

Display cellular tomography and segmentation

Trace microtubule

Compare single particle microtubule map to tomogram microtubule

Fit tubulin atomic into microtubule map

Microtubule atomic model

Atomic contacts between protofilaments

More Chimera capabilities


Tomography NetCDF file. The original tomography MRC map header claims it is signed 8-bit data. It is in fact 8-bit unsigned. Map reinterpretted as unsigned and inverted (0 becomes 255, 255 becomes 0) was saved in a NetCDF file That file also contains subsampled copies of the map with step size 2 and 4. Those were created with the Chimera VolumeProcessing/ script, opened as precomputed subsamples with volume viewer and then saved in the netcdf format. The subsamples allow quick display of the full map without having to read full map (~300 Mbytes) from disk.

Fitting with symmetry. Plugin allows fitting one copy of a molecule (e.g. tubulin dimer) by hand or with local optimization while other symmetric copies automatically update their positions. Good for seeing clashes.

Clash detection. Have atomic clash detection that can be used during fitting. Can show lines between molecule being fit and neighbors when atoms are close. Real-time update is currently (1.2382) slow even for two tubulin dimers (~6000 atoms x 2) because calculation is in Python.

Data sets

Cellular tomography. EM tomography (EMDB 1273) of stained plastic fixed human cytotoxic T lymphocyte in contact with a target cell. Examined transport of lytic granules to synapse along microtubules.

Stinchcombe JC, Majorovits E, Bossi G, Fuller S, Griffiths GM.
Centrosome polarization delivers secretory granules to the immunological synapse.
Nature. 2006 Sep 28;443(7110):462-5.

Microtuble density map. Microtubule single particle cryoEM construction to 8A resolution. (Obtained from KH Downing, not in EMDB as of May 2007). Publication proposes that single particle reconstruction is better than helical reconstruction for this data. Rises 3 monomer units in one turn in left-handed helix. This is 1.5 dimers and implies that the filament has a seam -- ie is not strictly helically symmetric unless alpha and beta monomers are considered identical. 13 protofilaments parallel the microtubule axis. At alpha and beta tubulin have 41% sequence identity and are essentially indistinguishable at 8A resolution. Microtububule is about 25 nm in diameter. Symmetry: z-translation 40.6 A monomer to monomer, rotation by angle 360/13 (~= 27.7 degrees) and translation by 3*40.6/13 (~= 9.37 A). I convolved 8A map with 10A Gaussian for simpler presentation.

Li H, DeRosier DJ, Nicholson WV, Nogales E, Downing KH.
Microtubule structure at 8 A resolution.
Structure. 2002 Oct;10(10):1317-28.

Tubulin atomic model. Atomic model of alpha and beta tubulin (PDB 1jff) at 3.5A obtained by electron crystallography of 2-D zinc-induced tubulin sheets. One GTP, one GDP, and one taxol ligand. Protofilaments are anti-parallel one another, unlike microtubule organization.

Lowe J, Li H, Downing KH, Nogales E.
Refined structure of alpha beta-tubulin at 3.5 A resolution.
J Mol Biol. 2001 Nov 9;313(5):1045-57.