Upton, New York
October 1, 2008
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Segments of the soybean mosaic virus, modeled from a
reconstruction of the virus structure using
cryo-electron microscopy, scanning transmission electron
microscopy, and X-ray fiber diffraction. These segments
are from computational modeling - they are based on an
image reconstructed from thousands of cryo-EM images.
Before the scientists could carry out the
reconstruction, they had to determine the helical
symmetry of the virus, which required data from X-ray
fiber diffraction and STEM. |
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Typical STEM image of unstained specimen used for
mass-per-unit-length measurement. Straight, thicker
particle is tobacco mosaic virus internal control.
Curved particles are soybean mosaic virus. |
Flexible filamentous viruses make
up a large fraction of known plant viruses and are responsible
for more than half the viral damage to crop plants throughout
the world. New details of their structures, which were poorly
understood, have been revealed by scientists using a variety of
sophisticated imaging techniques at the U.S. Department of
Energy’s Brookhaven National
Laboratory and collaborating institutions.
These findings, just published in the October 1, 2008, issue of
the Journal of Virology, may lead to new ways to protect crop
plants from viruses and other forms of damage. The structural
information may also benefit scientists interested in using
viruses as agents of biotechnology to coax plants to produce
other useful products, such as pharmaceuticals.
“These are very important viruses, and we knew almost nothing
about their detailed structure before these studies,” said
Gerald Stubbs, a structural biologist at Vanderbilt University
and lead author on the paper. “If you are to come up with any
molecular way of combating these plant diseases, you need to
know the details of their structures.” For example, structural
information could help scientists design molecules that
interfere with the virus’s ability to infect plant cells.
The scientists from Vanderbilt, Brookhaven, Boston University,
Illinois Institute of Technology, and the University of Kentucky
studied the structures of two plant viruses from unrelated
families, the Potyviridae and Flexiviridae, using a combination
of complementary imaging techniques — x-ray diffraction at DOE’s
Argonne National Laboratory, cryo-electron microscopy at
Vanderbilt, and scanning transmission electron microscopy (STEM)
at Brookhaven.
“Brookhaven Lab is home to one of only a few STEM machines in
the world,” said co-author Joseph Wall, a biophysicist at
Brookhaven who designed and runs the facility.
“These techniques are very complementary,” said Stubbs. “People
have been trying to get this structural work started for
decades, more than 40 years. It’s been very difficult and there
have been a number of obstacles, including the fact that it’s
very hard to make good samples of these viruses. But even after
we were able to do that, and analyze the structures using x-ray
diffraction and traditional electron microscopy, there were a
lot of ambiguities in the results. Those techniques gave us
several answers and we didn’t know which was correct.”
The STEM technique used at Brookhaven Lab provided the
definitive answer. Though by itself, STEM cannot determine the
structure of the viral protein coat, it is able to put
boundaries on the number of molecules in each “turn” of the
spiral-shaped structure. That is, it doesn’t tell you the shape
of the molecules but it can count them.
“STEM measures the number of electrons scattered from a length
of filament, thereby ‘weighing’ the segment relative to a
standard — in this case, tobacco mosaic virus,” said
Brookhaven’s Joe Wall. “Knowing the mass per unit length, the
subunit size, and the axial repeat gives the number of subunits
per turn.” According to Stubbs, “The number of molecules per
turn in the helix is the key. It allowed us to determine which
structure of the alternatives we’d come up with from other
techniques was correct.”
One surprise was that the two viruses the scientists studied,
though from unrelated families, turned out to have very similar
structures: thin filaments with a spiral structure featuring
just under nine molecular subunits per helical turn. “The spiral
is so tightly packed that it could not be seen by conventional
electron microscopy,” Stubbs said.
Some scientists had predicted there would be similarities
between these virus families, but there was a lot of
disagreement. This study provides the first experimental
evidence that they are indeed similar in shape. That could speed
up the pace of discovery because what scientists learn about one
family can be applied to the other.
Some potential applications include engineering molecules, based
on the viral structure, that interfere with the viruses’ ability
to infect plants. Another would be using modified versions of
these viruses to introduce into plants genes that help protect
the plants from viruses, or even from other forms of damage,
such as insect attacks. Still another application would be to
use modified viruses to introduce genes instructing plants to
make other useful products — for example, antibiotics or other
drugs.
This research was funded by the U.S. National Science Foundation
and the U.S. Department of Agriculture, with funding for several
of the research facilities provided by the offices of Basic
Energy Sciences (BES) and Biological and Environmental Research
(BER) within DOE’s Office of Science, and by the National
Institutes of Health. The STEM facility at Brookhaven Lab is
partially funded by BER and by Fee-for-Service support.
For information about fees, contact Joseph Wall,
wall@bnl.gov or go to:
http://www.biology.bnl.gov/stem/stem_charges.pdf.
For additional information about STEM, go to:
http://www.biology.bnl.gov/stem/stem.html.
One of ten national
laboratories overseen and primarily funded by the Office of
Science of the U.S. Department of Energy (DOE), Brookhaven
National Laboratory conducts research in the physical,
biomedical, and environmental sciences, as well as in energy
technologies and national security. Brookhaven Lab also builds
and operates major scientific facilities available to
university, industry and government researchers. Brookhaven is
operated and managed for DOE’s Office of Science by Brookhaven
Science Associates, a limited-liability company founded by Stony
Brook University, the largest academic user of Laboratory
facilities, and Battelle, a nonprofit, applied science and
technology organization. |
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