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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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