Causes of Life

For biologists, x-ray crystallography has always been a tricky technology. Harder than getting a good beam was growing large crystals of biological molecules-a task that's been compared to building regular structures from wiggly bits of Jello.

Today, synchrotron light from facilities such as Berkeley Lab's Advanced Light Source may make it possible to use protein crystals as small as 50 microns (50 millionths of a meter) in length.

The crystals themselves may also become easier to grow, thanks to a unique robotic system designed and built by Joseph Jaklevic, head of Engineering Sciences, and his colleagues in the Engineering Division's Bioinstrumentation Department.

"The idea for a high-throughput combinatorial approach to crystal growth came from Peter Schultz," says Jaklevic. "The basic idea is that, instead of having to plod through all the hundreds of ways you might get a protein to crystallize, you more or less try 'em all at once."

Schultz pioneered combinatorial methods as a member of the Lab's Materials Sciences Division; he recently became head of the Novartis Institute for Functional Genomics in La Jolla, California. He and his colleague Raymond Stevens of the Lab's Physical Biosciences Division saw the combinatorial approach as a natural solution to the challenge of growing protein crystals.

That's because "biologists really have no idea what the best conditions are for growing crystals of a new protein," says Derek Yegian, a member of the team that built the new robotic system. "Different proteins precipitate out of solution and grow at different rates-or don't grow at all-depending on the solution's acidity, temperature, concentrations of salts, and lots of other variables. "

The innovative robot above, designed and built by Joe Jaklevic and his colleagues in the Engineering Division's Bioinstrumentation Department, can automatically grow crystals of a novel protein by screening 480 different growth solutions at once.

Only the very purest proteins will crystallize, and pure protein is expensive; even common commercial proteins can cost hundreds of dollars a gram. Often hundreds of combinations of variables must be tried before a novel protein can be crystallized from solution.

Most trial solutions are prepared by hand at the rate of about 30 an hour, typically requiring one to 10 microliters of pure protein for 50 to 100 "coarse-screening" trials; whether a particular solution yields a crystal is apparent only days or weeks later.

"Manual methods are slow and error-prone," says Yegian, and although some steps have been automated within the past few years, "commercial robots are not much better." With the Bioinstrumentation Department's new robotic system, however, once a target protein has been chosen, 480 different variations of growth solution can be coarse screened all at once, each in its own tiny reservoir.

A set of trials starts with 10 empty, transparent plastic cassettes. As each cassette is loaded from a stacker, the robot lifts its lid, and 48 needles simultaneously coat the lips of the 48 tiny bowl-shaped wells inside with a thread of grease.

Next the wells are half-filled with growth solution, called the "mother liquid," by syringes fed by banks of cylinders; each bank of 10 holds 48 different solutions, varying by types of salts, buffers, and so on.

Meanwhile, at a separate station, a syringe deposits a mixture of the protein and the appropriate mother liquid on each of 48 circular transparent cover slips. As the cassette is stepped through, each row of eight slips is inverted and sealed over the corresponding wells. The robot needs six minutes to set up and seal the 48 cover slips for a cassette, which sets

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