Imagine going into a cell and watching the intricate writhing of a ribosome as it cranks out vital proteins. Structural biologists are nearing such powers of observation with new analytical techniques that manipulate ribosomes and other macromolecules while tracking changes in their structure and shape.

Among the most remarkable of the new methods is three-dimensional cryo-electron microscopy (cryo-EM). In the hands of researchers such as HHMI investigator Joachim Frank at Health Research Inc. at the Wadsworth Center in Albany, New York, cryo-EM has proven to be one of the few techniques capable of visualizing large, dynamic molecules. "Cryo-EM's strength is that it allows a three-dimensional reconstruction of macromolecules that cannot be crystallized for analysis by x-ray crystallography," Frank says. The technique also makes it easier and faster to explore how such dynamic molecules change shape as they carry out their biological functions. "In this way," he says, "we can essentially produce a movie of the macromolecule in action, which would be much more difficult and time-consuming to generate using other methods."

Here's how cryo-EM works. First, Frank and his colleagues immerse their macromolecules—let's say ribosomes—in a water solution. Then, they flash-freeze the molecules in supercold liquid ethane. The rapid freezing imprisons the ribosomes in ice, preserving their original conformation. Next, using an electron microscope with a low-intensity beam to avoid damaging the molecules, the scientists obtain images of thousands of the captive ribosomes. They then use sophisticated software called SPIDER, developed in Frank's laboratory, to transform these low-contrast, "noisy" images into a detailed, three-dimensional map of a ribosome.

Cryo-EM's power to detect the contortions of working macromolecules was highlighted this past July in a report by Frank and his colleagues in the journal Nature.The researchers announced that they had detected a subtle ratcheting rotation deep inside the ribosome—the complex biomolecule "powered" by ribonucleic acid—at a key stage in the protein-building process. In their paper, the scientists characterized the ratcheting as a rapid rotation of one of the ribosome's two subunits. This rotation occurs just as the messenger RNA (mRNA) and its two attached amino-acid-carrying transfer RNA (tRNA) molecules are advanced. Their analysis revealed that at a key point, the smaller subunit (called 30S) rotates about six degrees with respect to the larger subunit (50S). Then, after a chemical reaction advances the mRNA-tRNA complex, the smaller subunit rotates back. In this herky-jerky manner, the two subunits cooperate to build the long chains of proteins.

Discovery of the movement offers yet another clue to how the ribosome creates the enzymes and other components of the cell's machinery. "There have been hypotheses about subunit movement in the ribosome for years," Frank says, "but there has never been a direct confirmation of this. The problem was that all the evidence was indirect. And only now, with cryo-EM, can we visualize the ribosome with such clarity."

To Frank, the achievement underscores the broader promise of cryo-EM. "The niche for cryo-EM is likely to be pretty big," he says, "because it can be used to study macromolecular machines, like the ribosome, in all their different states, with all their necessary functional materials attached, as they normally operate in the cell." He emphasizes, however, that the far higher resolution achievable with x-ray crystallography makes the two analytical techniques partners rather than competitors. "It's not that x-ray crystallography puts cryo-EM out of business," he says. "On the contrary, it really puts it into business. By marrying these two techniques, one can proceed from the detailed atomic structure, from crystallography, to make sense of the different conformation you see with lower-resolution cryo-EM."

—TB

Photo: Christopher Denney

Download this story in Acrobat PDF format.
(requires Acrobat Reader)

Reprinted from the HHMI Bulletin,
January 2001, Pages 12-17
©2001 Howard Hughes Medical Institute