Cryo-EM is a way to view protein structures—now at atomic resolution—as they do their thing in the biological world.

This three-dimensional cryo-EM image of a rotavirus enables researchers to infer the role of two proteins on the virus's temporary, outermost coat. The VP4 spike protein (red), which helps the virus puncture the membranes of target cells, is held in check by the VP7 protein (gold) until the virus is primed for infection. In green is a third protein, VP6, found in the middle coat.

For an electron microscope, which needs a vacuum to clear the air, water is the enemy. “It contaminates the sample, makes the vacuum dirty, and essentially destroys the experiment,” says Nikolaus Grigorieff, an HHMI investigator at Brandeis University. “Of course, in biology, water is the one substance that is present everywhere.”

The solution is to flash-freeze the sample and use the electron microscope, one of the most sensitive tools in science, to look at proteins or other biological structures trapped in a frozen matrix. In electron cryomicroscopy, or cryo-EM, researchers freeze a sample in liquid ethane so quickly that the watery environment becomes a glassy, vitreous solid before it can spoil the experiment or crystallize into regular ice. The living structure is preserved whole, suspended in time.

The technique has recently begun to achieve the degree of sensitivity that makes it useful for structural biologists who want to resolve molecular structures at the atomic level. Increasingly, cryo-EM is seen as a natural complement to—and sometimes a substitute for—gold-standard methods such as x-ray crystallography for studying proteins, nucleic acids, and the complexes they form inside cells.

Grigorieff is a specialist in the field of cryo-EM. Working with fellow HHMI investigator Stephen Harrison of Harvard Medical School and Children's Hospital, Boston, he recently reported a structural map of an outer-coat protein locked in place on an intact rotavirus particle (see Upfront, “Piecing Together Rotavirus's Unique Approach”). The picture was not as detailed as Harrison's images of the protein captured by x-ray crystallography, but it allowed the scientists to visualize the protein's placement on the intact particle and infer its role in rotavirus infection.

For almost any technique in structural biology, the trick is to achieve high resolution without losing the fuller picture of how a structure looks and behaves inside a cell. That's a big challenge for x-ray crystallographers, who must isolate, purify, and coax living structures into an artificial crystalline lattice to study them. For one thing, crystallization is not always possible, especially for large structures made of many interlocking molecules. In addition, the crystallized form captures only one state of the structure: cellular parts are made to move, and locking them into a static position gives only one part of the story. Other structural biology techniques are limited in that they require dehydration, dyes, or harsh fixatives that can disrupt fragile chemical bonds.

Developed in the 1970s, cryo-EM has the advantage of being able to preserve molecular structures in multiple states and hence to give a more complete picture of a macromolecular machine in action.

Illustration: James Chen, Ethan Settembre, Scott Aoki, Xing Zhang, Richard Bellamy, Phillip Dormitzer, Stephen Harrison, and Nikolaus Grigorieff

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