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Brookhaven scientists and lead authors, from left, Marc Allaire, Allen Orville, and Alexei Soares in the National Synchrotron Light Source II (NSLS-II) ring building, now under construction at Brookhaven Lab. The research group’s work on acoustic drop ejection could improve macromolecular crystallography studies at the new facility. Photo: Brookhaven National Laboratory
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Brookhaven National Laboratory researchers are using
high-frequency sound waves in conjunction with extremely bright X-rays to get a
look at the atomic structures of the complex biological molecules that make our
bodies work.
Like a high-speed X-ray camera, a technique called
macromolecular crystallography provides researchers with 3D pictures of the
arrangement of atoms in molecules ranging from enzymes to nucleic acids. This
is done by making a crystal comprised of many copies of the particular molecule
and then bombarding it with beams of high-intensity X-rays that diffract, or
bend, as they interact with the electrons in the atoms of the molecule. These X-rays
then hit a detector, and are analyzed with a computer program to determine the
atomic-level image. Knowing the molecule's structure provides information about
its function, which may lead to important clues about how to create effective
drugs to prevent or treat a disease.
"X-ray crystallography has transformed our understanding of
biological processes," says Photon Sciences Directorate biophysicist Marc
Allaire. "Crystallographers working at Brookhaven's National Synchrotron Light
Source have determined the structures of numerous molecules, including those
from organisms responsible for the common cold, Lyme disease, and AIDS, in
addition to investigating how plants respond to environmental changes."
With a new class of brighter X-rays produced at next-generation
light sources like Brookhaven's soon-to-be National Synchrotron Light Source II
(NSLS-II), scientists will be able to study crystals measuring only a few
micrometers along an edge (like those from membrane proteins)—an ability that’s
not possible with conventional macromolecular crystallography.
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(a) Stroboscopic photomicrograph of a single 2.5 nL water droplet launched via ADE from the liquid surface. (b) Image from NSLS beamline X25 of the insulin microcrystal slurry supported on a micromesh (25 μm by 25 μm grid pattern) used to determine the structure. The 20 μm by 20 μm X-ray beam is centered at the intersection of the white cross hairs. (c) Illustration of the concepts for the raster-scanning X-ray diffraction strategy with a microdiffractometer and a several-micrometer-wide X-ray beam. Image: Brookhaven National Laboratory
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But there's a problem: As the crystal size is reduced, the
diffraction signal weakens due to noise from the surrounding solvent. To
address this technological gap, scientists from Brookhaven and Labcyte Inc. (Sunnyvale, Calif.)
are incorporating a technique called acoustic drop ejection (ADE) into studies
at NSLS and the Advanced Photon Source (APS) at Argonne National Laboratory.
The technique uses intense sound waves to launch from a solution of very small
droplets containing even smaller protein crystals through the air and to a
mounting mesh. There, the crystals are held in the line of X-ray beam.
"The idea that sound waves of great intensity near the surface
of a liquid will eject droplets was demonstrated in the 1920s," says Brookhaven
biologist Alexei Soares, a beamline scientist at NSLS for the Macromolecular
Crystallography Research Resource (PXRR). "Nowadays, ADE is used to transfer
living cells and isolated DNA without inducing strand breaks, suggesting that
it might be gentle enough for protein crystals. The possibility of using ADE to
move droplets of extremely small volume is ideal in minimizing the solvent
surrounding microcrystals."
First, the scientists had to make sure that the ultrasonic sound
waves—which have a greater frequency than we can hear—produced by ADE don’t
damage the crystals. The researchers grew two slurries containing solvent and
hundreds of microcrystals of insulin or lysozyme—each crystal measuring about
10 micrometers on a side, about the size of a single cell. With an ADE device
built by Labcyte, the group focused acoustic waves through the slurries, the
pressure of which eventually expels 2.5 nL drops of the crystal-filled liquid
to the mounting mesh.
The individual microcrystals, which are nearly impossible to see
even with powerful microscopes, were located within the drops by scanning a
very small X-ray beam across the micromeshes. Then, X-ray diffraction was
performed at NSLS and APS to determine the crystal structures.
"We were astounded by the results. The fragile microcrystals are
not harmed by the novel ADE mounting technique, and therefore they produce
high-resolution X-ray structures," says Brookhaven biologist and PXRR beamline
scientist Allen Orville. "These developments enable new opportunities in
automated microdiffraction and the manipulation of microcrystals."
Their results were published in Biochemistry.
Because ADE is able to transfer an ejected drop to a precise
location, the researchers also foresee the possibility of using the technique
to load multiple drops of different crystal solutions on a single mesh,
speeding up the discovery process.
"Using ADE, a new strategy could be developed for
high-throughput structural screening of microcrystals against libraries of
chemical compounds for drug development," Allaire says.
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