The MAD Method
A beamline that shunts off the main storage ring of Berkeley Lab's ALS carries a finely focused beam of x-rays hurtling at near light speed towards a target inside an experimental station.
Courtesy Lawrence Berkeley National Laboratory
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Until the early 1990s, a technique called isomorphous replacement was the solution to the phase problem. "You start off by putting your protein crystal into a beam and you collect all data at various angles, which is called a data set," Kappler explains. "Then you collect additional data from crystals soaked in heavy metals, like mercury, platinum or uranium salts, that will bind to very specific places on the protein, and you can use those data to calculate your phases. You get the same spots you got before, in the same positions, but the intensity changes. Some get duller, some brighter. That change of intensity tells you something about the phase of the x-rays.
"It's a very laborious technique and it's hit or miss. You may have gotten a beautiful protein crystal that diffracts very well in an x-ray beam. You get a beautiful data set, and you still may spend several years trying to get a derivative with one of these heavy metals that gives you a second data set that you can use to get phase information."
The MAD method replaces the heavy metals with a single atomic substitution in the crystal: Most often, the amino acid methionine is replaced with selenomethionine, which is chemically very similar. The resulting protein structure is essentially identical, but special scattering from selenium atoms is sufficiently strong to be used to determine the phase of the x-rays by simply tuning the x-ray wavelength.
"In MAD," says Hendrickson, "instead of doing the chemistry of adding heavy atoms and changing structure, we can do the equivalent exchange by physics. We change the scattering properties by tuning the x-ray source. We can do all those measurements on one single sample, but without a tunable source of x-rays this experiment isn't feasible."
Using MAD, the average time for getting a protein structure might drop from years to weeks. Add to that the other advantages of a synchrotron x-ray source and suddenly crystallography is transformed from a grueling enterprise mastered by few to a technique that is no more challenging than other state-of-the-art tools. In short, with MAD and a synchrotron for the x-rays, biologists can obtain structures of protein assemblages larger than they'd ever imagined, they can do it with nearly single-atom resolution, and then they can gather all the information necessary for obtaining a structure within a day or two. "That's how impressive and powerful this technique is," says Kappler.
