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A Suspect Jackknife

Though scientists have known for many years that integrins are important in many physiological processes, detailed structural information on these molecules has been elusive.

The size of the integrins, and the fact that they span the membrane confounded structural studies of the proteins. In fact, the only way to solve the structure was to chop off the membrane-spanning regions and solve the individual parts separately by x-ray crystallography.

But until recently, there were no high-resolution structures even of these extracellular domains. Then the Arnaout group published the crystal structure of the domains in the journal Science about a year ago.

However, this structure showed that the ligand-binding head region was bent back, like a jackknife, to the point where it was almost touching the region of the protein that would connect the transmembrane "threads".

"The crystal structure provided a lot of new insight," says Adair, "But it does not seem that this 'jackknife' form is the major conformation for the intact molecule."

How the Technique of Electron Microscopy Works

The first electron microscope was built by Ernst Ruska in 1933, for which he received the Nobel Prize in 1986 at age 80. Electron microscopes use magnetic lenses to bend a beam of electrons to image tiny objects, similar to the bending of light by glass lenses in a light microscope. EM looks at a range of magnifications, from no more than an ordinary light microscope that magnifies up to 60 times to those that magnify up to 1,000,000 times.

TSRI is one of the few centers in the world with an integrated program in electron microscopy of biological complexes and macromolecular machines. The Center for Integrated Molecular Biosciences is directed by Ron Milligan. Two other Scripps scientists, Bridget Carragher and Clint Potter, were recently awarded an NIH Research Resource Grant to develop automated molecular microscopy. Adair and Yeager used the Philips/FEI microscopes at CimBIO to collect their data.

Cryo-EM, which is the technique used in the current study, requires that samples be spread in a thin film and then frozen on a copper meshwork grid. The freezing process occurs in a few milliseconds at about a million degrees a second. In this way the frozen water is in a glass-like vitreous state, which is an excellent environment to preserve biological molecules in near-physiological conditions—a significant advantage over x-ray crystallography, where the proteins are often crystallized in pieces and in exotic buffers.

Adair and Yeager purified the integrin molecules from human platelets in mild detergent solutions that mimic the oily environment of the platelet membrane.

The computational challenge was to sort out thousands of different views of the integrin molecules and combine them to derive a 3-D map. The map revealed the overall shape and size of the entire integrin, including the large extracellular domain, the small cytoplasmic domains and the transmembrane coiled-coil.

Adair and Yeager then used the EM structure as a "molecular envelope"—like a mold, into which the 12 domains derived by x-ray crystallography could be docked. By this combined approach a detailed description of the structure and action of complicated molecular machines such as integrins can be derived.

The article, "Three-dimensional model of the human platelet integrin alphaIIbbeta3 based on electron cryomicroscopy and x-ray crystallography" is authored by Brian D. Adair and Mark Yeager and appears in the October 29, 2002 edition of the journal Proceedings of the National Academy of Sciences.

This work was supported by the National Institutes of Health, the National Heart, Lung, and Blood Institute, and a postdoctoral fellowship from the California affiliate of the American Heart Association (to Adair). During the course of this work, Yeager was an Established Investigator of the American Heart Association and is now the recipient of a Clinical Scientist Award in Translational Research from the Burroughs Wellcome Fund.

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Surface-shaded 3D density map of the alphaIIbbeta3 heterodimer at 20 Ångstroms resolution determined by electron cryomicroscopy and computer image analysis. The large ectodomain (blue, violet and orange) and small cytoplasmic domain (red) are connected by a rod of density (yellow) that is interpreted as two parallel transmembrane alpha-helices. A 30 Ångstrom thick box indicates the putative position of the membrane bilayer.