With his electron cryotomography (ECT) technique, Grant Jensen, HHMI investigator at the California Institute of Technology, is drilling down to the molecules of life. The technique is giving him and his colleagues the visual access to biomolecular complexes that do things like dump waste across cell membranes, orchestrate gene expression, and kill other cells.
A central step in ECT, the “cryo” part, is to plunge-freeze specimens in liquid nitrogen. This prevents the evaporation of cells’ liquid interior that would otherwise occur in an electron microscope’s vacuum sample chamber. Air molecules are sucked out so they don’t get in the way of the microscope’s electron beam, which can illuminate samples on finer scales than can light in an optical microscope. The flash-freezing step locks the water in place, preventing the sample from drying up. It allows Jensen to examine the protein machinery inside of cells in a more natural, hydrated state than is possible with conventional electron microscopy.
“Seeing” the molecular machinery of life requires more than that, however, which is where the tomography part of ECT comes in. “If you can take a picture of an object from a lot of different directions, you can reconstruct what the three-dimensional structure must be,” says Jensen. He and his colleagues take about 100 pictures, tilting the specimen about 1 degree between each. To Jensen, this amounts to a CAT scan for protein complexes. “We are getting 3-D images of cells in near-native state [cellular water in place] to molecular resolution [2–4 nanometers],” says Jensen.
One of Jensen’s favorite examples is the visual clarity that ECT provided for a bacterial weapon system his group described in the February 2012 issue of Nature. The system is known somewhat misleadingly as a type VI secretion system, inside of Vibrio cholerae bacteria cells. “To our surprise it looked like a big cannon inside the cells that was linked to the membrane. Imagine a harpoon inside a barrel, but the barrel contracts and acts like a spring,” he says. When a Vibrio cell bounces into another cell, such as an Escherichia coli bacterium, the spring contracts and the harpoon drives forward into the E. coli. “Just by seeing the pictures—this thing in a loaded and extended form, and in a contracted fired form—it immediately became clear what was going on.”