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
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. "
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
"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
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 the rate of the entire coarse-screening preparation process.
The cassettes are automatically stored at
constant temperature after filling. Water slowly
diffuses from the hanging drop into the mother
liquid in the reservoir, and the concentration of
protein increases. Crystals precipitate in some of
"The smaller the drop size, the quicker the
crystallization," Jaklevic explains. "We're trying to
get crystallization with a few nanoliters of protein instead of microliters-a reduction by a
factor of a thousand. We will see crystals within a few hours to a few days."
A high-resolution ccd camera checks the cassettes twice a day and detects the growth
of even tiny crystals in the transparent reservoirs; reservoirs showing crystals can be
returned to the sample preparation unit to begin a second cycle, under conditions finely
tuned for optimum growth. Solutions that don't produce good results are rejected. The
preliminary system has been successfully tested for reliability, consistency, and speed on
commercially available proteins and on novel proteins from the laboratories of Schultz and
Stevens. It includes hardware built from scratch or adapted by the Bioinstrumentation
Department and uses original software to control the numerous delicate steps in the
operation; Jaklevic and his colleagues are working to replace or improve some
off-the-shelf components. Before long, they hope to see the full system in regular use at
the Advanced Light Source and elsewhere.
Jaklevic and his department have
designed, built, and modified numerous
ingenious robots used in the life sciences,
including devices for the Lab's Human
Genome Center credited with proving
several years ago that prodigious rates of
automated gene sequencing were possible
with no loss of accuracy. Recently the
Bioinstrumentation Department built the
second-generation microdot arrayers used
by Life Sciences Division researchers to
study gene expression in heart disease.
"To accommodate the enormous growth in
the biological sciences at Berkeley Lab we need to exploit the Lab's traditional
multidisciplinary strengths," says Jaklevic. "We hope biologists will see how much they
can benefit by making use of engineers. We have a lot of engineers here who can 'speak
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