The Synchrotron Advantage
By developing a technique for fine-tuning x-rays, HHMI investigator Wayne Hendrickson reduced from years to weeks the amount of time necessary for solving protein structures.
Photo: Christopher Denney
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"If you're going to understand function, the structure is an enormous help," says HHMI investigator Brian Matthews, a structural biologist at the University of Oregon. "There are two ways to determine structure. One is by nuclear magnetic resonance and the other is by x-ray crystallography. NMR is most powerful for smaller proteins that can't be crystallized. For larger complexes and higher resolution images there is no substitute for x-ray crystallography."
X-ray crystallography was invented before World War II, and its promise in biology was demonstrated first in the 1950s when the Nobel laureates Max Perutz and Sir John Kendrew used it to solve the structure of the oxygen-carrying proteins myoglobin and hemoglobin. For the next 30 years, researchers relied on low-power, single-frequency x-ray sources in their labs to eke out the information they needed to decipher the structure of a protein. X-ray crystallography was difficult, and remained the tool of a limited number of specialists.
The switch to synchrotron x-rays—and the attendant democratization of the technique—was a slow one, beginning in 1971 with the work of European investigators on a makeshift beamline at a synchrotron in Hamburg, Germany. Synchrotron radiation brought with it a host of advantages. For starters, the synchrotron x-rays were 1,000 times more powerful than the x-ray sources found on smaller machines located in academic crystallography centers. This increased radiation intensity meant that investigators could collect data more quickly. Synchrotron radiation could also be focused in such a way that all the energy in the beam could be brought to bear on a very small crystal. "It means you can measure data from much smaller samples than was the case before," says Matthews.
But there were problems, too. The intense energy in an x-ray beam can damage a protein crystal. The more potent the beam of x-rays, the faster the protein crystals will decompose. But researchers developed methods of freezing crystals that allowed the precious crystals to withstand the x-ray assault. "It used to be that if you froze the crystal, ice particles would form within the protein crystal and that would destroy and disorder it," says Matthews. "But if you can use a cryoprotective solution, like anti-freeze for your car, and you freeze the crystal very rapidly, it maintains the three-dimensional order. Now you can continue to collect data from that crystal for very long periods, even in a synchrotron beam. It means you can collect a lot of data from a single crystal, even a very small one, sufficient data to determine the whole structure."
Perhaps the biggest advantage offered by synchrotrons is that the beams can be tuned to multiple wavelengths, which is not the case with laboratory x-ray sources. This allows researchers to use a technique called MAD, for multi-wavelength anomalous diffraction, to take on the most difficult challenge in crystallography—the phase problem.
"X-rays basically go in straight paths," says Hendrickson, who developed MAD. "You don't have any lenses, so you can't form images directly. You need to do image formation by calculation, and that is not a trivial process." Two kinds of information are needed for the calculation. One is the intensity at each point in the diffraction pattern that forms as x-ray waves from the crystal hit the detector plate. The other is the phase, which is the relative excursion within a wavelength along the path for each of the various x-ray waves. That information is not inherent in the x-ray pattern.
