When Gang Ren whirls the controls of his
cryo-electron microscope (cryoEM), he compares it to fine-tuning the gearshift
and brakes of a racing bicycle. But this machine at the U.S. Department of
Energy (DOE)’s Lawrence Berkeley National Laboratory (Berkeley Lab) is a bit
more complex. It costs nearly $1.5 million, operates at the frigid temperature
of liquid nitrogen, and it is allowing scientists to see what no one has seen
At the Molecular Foundry, Berkeley Lab’s
nanotechnology research center, Ren has pushed his Zeiss Libra 120 Cryo-Tem
microscope to resolutions never envisioned by its German manufacturers,
producing detailed snapshots of individual molecules. Today, he and his
colleague Lei Zhang are reporting the first 3D images of an individual protein
ever obtained with enough clarity to determine its structure.
Scientists routinely create models of proteins
using X-ray diffraction, nuclear magnetic resonance, and conventional cryoEM
imaging. But these models require computer averaging of data from analysis of
thousands, or even millions of like molecules, because it is so difficult to
resolve the features of a single particle. Ren and Zhang have done just that,
generating detailed models using electron microscopic images of a single
He calls his technique “individual-particle electron
tomography,” or IPET. The work is described in PLoS One.
The 3D images reported in the paper include those
of a single IgG antibody and apolipoprotein A-1 (ApoA-1), a protein involved in
human metabolism. Ren’s goal is to produce individual 3D images of medically
significant proteins, such as HDL—the heart-protective “good cholesterol” whose
structure has eluded the efforts of legions of scientists armed with far more
powerful protein modeling tools. “We are well on our way,” says Ren.
Ren has the credentials of one who knows what he
can do. He was recruited to work at Berkeley Lab in August 2010 from the University of California
at San Francisco,
where he had used a cryo-electron microscope and more conventional averaging
techniques to discern the 3D structure of LDL—the “bad cholesterol” thought to
be a major risk factor for heart disease.
His images of single proteins are a bit fuzzy, even
after they are cleaned up by complex computer filtering, but very informative
to the trained observer. These individual particles are extraordinarily tiny,
requiring Ren to zero in on a spot of less than 20 nm. He has reported protein
images as small as 70 kDa. That’s kilodaltons, a Lilliputian scale (expressed
in units of mass) set aside for taking the measure of atoms, molecules, and
snippets of DNA. It’s a more useful way to size soft objects like proteins that
can be clumped, stringy, or floppy.
Unlike the sculptural images of protein models, a
suite of these photographs can convey a sense of these particles in all their
nanoscale floppiness. Within the complex structure of these proteins lies the
secrets of their function, and perhaps keys to drugs that block the bad ones
and promote the good ones. With some additional computer filtering, a
high-contrast model of protein can be generated from the images and animated to
show its moving parts in 3D.
“This allows you to see the personality of each
protein,” says Ren. “It is a proof of concept for something that people
thought was impossible.”
By observing the structure of single proteins, it
is possible to understand their flexible, moving parts. “This opens a door for
the study of protein dynamics,” Ren says. “Antibodies, for example, are not
solid. They are very flexible, very dynamic.”
How did Ren coax so much versatility out of his
Libra 120? “It’s not a very high-end model,” he concedes. Much has to do with
the accessories he bolts on to the machine, and with his own artistry and
patience. He’s equipped the microscope with a $300,000 CCD camera, some
powerful image-processing software, special contrasting agents, and a device
called an energy filter that sifts through the digitized camera data and
culls weak signals. Thoroughly familiar with his customized machine, he also
employs an element of elbow grease, working long hours to draw out the powerful
images from a torrent of digital noise.
The multiple angles used to create the 3D portrait
help resolve the faint molecular image. “All images are noisy,” Ren explains. “In physics, the noise is inconsistent among the images, but the signal—the
object or protein—is consistent. By using this approach, we find the consistent
portion (the signal) can be enhanced, while the inconsistent portion (the
noise) will be reduced substantially.”
Electron microscopes focus streams of electrons
rather than light to see incredibly tiny things. The short wavelength of an
electron beam enables much higher resolution and magnification than visible
light. Powerful electron microscopes have been used for decades to probe
materials at atomic scale; and right next door to the Molecular Foundry is
Berkeley Lab’s National
Center for Electron Microscopy.
The TEAM 0.5 microscope can distinguish objects as small as the radius of a
hydrogen atom. But these heavyweight microscopes pull off this atomic-scale
resolution with pulses of energy that would obliterate most soft biological
proteins. The high power electron microscopes are used primarily for probing
atomic structure of strong, solid materials, such as graphene.
Ren’s laboratory specializes in cryoEM, which examines
objects frozen at -180 C (-292 F). A bath of liquid nitrogen flash-freezes samples
so quickly that no ice crystals form. “It is amorphous, like glass,” Ren says.
The protein samples are frozen on a disk the size of baby’s fingernail, filled
with tiny wells 2 um across. The disk is inserted into the microscope on a
rotating support that can tilt the sample up to 140 degrees inside a vacuum—sufficient
camera angles to produce a 3D perspective. “The challenge is to isolate it from
the air, and to turn it without vibrations, even the vibrations from the
bubbling of liquid nitrogen,” says Ren.
The extremely low temperature fixes the samples and
prevents them from drying out in the vacuum needed for the electron scan. It
creates conditions favorable for imaging at much lower doses of electrons—low
enough to keep a single soft protein intact while more than 100 images are
taken over a one-to-two hour period.