Beta-lactamase disarms penicillin, breaking it down before it can do its antibacterial work. But the beta-lactamase inhibitor protein, BLIP, interferes, paving the way for penicillin to do its work.
Exactly how is no longer a mystery. The complex of beta-lactamase and BLIP was solved, painfully, long ago. “It took Natalie Strynadka”—now at the University of British Columbia—“a couple of years to solve,” says Randy Read, professor of hematology at the University of Cambridge and lead developer of Phaser, a structural biology software tool. “Today it's something that can be solved by one of Phaser's tutorials in five minutes.”
One problem with the old, painful methods, was signal-to-noise. Researchers had already solved the structures of both molecules and they could find the beta-lactamase in the crystal of the complex. But that protein, being twice as large as it's inhibitor, overshadowed the tiny BLIP.
Phaser, however, uses probabilistic methods to keep track of what the beta-lactamase component has already explained in the data. “All we have to do is explain what's unexplained by orienting the BLIP component,” says Read. “It increases the signal-to-noise ratio considerably.”
This concept underlies Phaser, which integrates with both CCP4 and Phenix, and uses statistical methods to select and place models for solving structures by molecular replacement. These methods provide more sophisticated algorithms than a black and white selection or rejection of models that agree well or do not. “We're able to deal in a nice, smooth way with models of different quality,” says Read. As a result, Phaser allows the solution of structures with poorer models than possible with older technology.
Phaser was recently put to work in an SBGrid project that employed its grid computing resource to search the protein database and try thousands of different models to find the best possible starting point. This procedure, published in 2010 in the Proceedings of the National Academies of Science, was effective but slow. “It required that the full set of calculations be run on every possible choice in the database even if the first choice gave a clear solution,” says Read. The latest version of Phaser recognizes the first clear solution immediately.
There are many approaches to using Phaser and many possible stumbling blocks. For this reason, Read maintains the Phaser Wiki (http://www.phaser.cimr.cam.ac.uk/index.php/Phaser_Crystallographic_Software), which includes a FAQ, troubleshooting information, and a section where users can share their own success stories and strategies for using Phaser.
As an example, Read himself recently used Phaser in a creative way to solve the structure of human angiotensinogen, a protein involved in regulating blood pressure. The human angiotensinogen electron density maps were not good enough to finish a complete model. “We got hold of rat and mouse angiotensinogens and developed some new tricks to use the density from one crystal to solve the structure in another crystal,” says Read. The process, which required sophisticated knowledge of Phaser, is something Read is folding into the program for anyone to use automatically.
This is the beauty of technologies as they mature. “The expertise gets concentrated in the instruments and in the people who make programs for the instruments,” says Read. “That frees up the people who use those techniques to become sophisticated in something else.”
– Elizabeth Dougherty