Even a workhorse can't do everything. The dual
advantages of high resolution and fast throughput
make synchrotron-based x-ray crystallography, like
that which is being carried out at Berkeley Lab's
Advanced Light Source, the undisputed mainstay for
solving protein structures. However, some 20 to 40
percent of all proteins are extremely difficult or even
impossible to crystallize, including many found in the
membranes which control the transportation of
molecules and communication of signals across cell
surfaces. This means that other technologies will also
have critical roles to play. One alternative that does
not require crystallized proteins is nuclear magnetic
resonance (nmr) spectroscopy, a technology that
exploits the spin of certain atomic nuclei to obtain
structural, spatial, and even dynamic information
about those nuclei. Conventional nmr spectroscopy,
however, is essentially limited to the study of small
proteins. For the study of macromolecular protein
complexes, especially those that are difficult to coax
into crystals, the best alternative to x-ray
crystallography may be electron microscopy.
Seeing the bigger picture
The beam of an electron
microscope can produce
diffraction patterns from
arrays rather than the
required for x-ray diffraction.
In the case of membrane
proteins, these 2-D crystals
can be created in a more
natural setting-sheets of
fatty molecules called
lipids-than the heavy
concentrations of detergents
needed to form 3-D crystals.
crystallography is the only
way to go, as was the case
two years ago when
biophysicists Eva Nogales,
Sharon Wolf, and Kenneth
Downing, of Berkeley Lab's
Life Sciences Division,
announced the completion of
the first 3-dimensional atomic
model of a protein called
"tubulin," a highly versatile
molecule which, among other
functions, enables a cell to
undergo mitosis. At a
resolution of 3.7 angstroms,
this model provided the first
highly-detailed three-dimensional look at tubulin, including the site where the protein
interacts with the anti-cancer drug taxol.
The use of electron-based rather than x-ray-based crystallography techniques was
crucial to the creation of this model. As Downing explained, "obtaining diffraction patterns
with an electron beam enabled us to work with crystals only one molecule in thickness,
giving us our high resolution."
Solving tubulin's atomic structure was a spectacular success-scientists had been trying
to do it since the protein's discovery in the 1960s-but the project took the Berkeley Lab
researchers more than six years to complete. During that time, they recorded some 4,000
images and electron diffraction patterns which Nogales processed to come up with the
159 images and 93 diffraction patterns used to do a computer reconstruction for the final
3-D tubulin model.
The technique used to solve the tubulin structure is called cryo-electron microscopy or
"cryo-em" because the images were recorded with an electron microscope equipped with
a "cold stage." Freezing the protein preserves it in its native hydrated state and helps
protect it from radiation damage. Another addition to electron microscopy with
exceptionally strong potential for solving the structures of macromolecular protein
complexes is a technique called "single-particle image analysis."
In single-particle image analysis, thousands of
images of randomly-oriented individual protein
molecules are recorded via an electron
microscope. A computer is then used to align
these thousands of randomly-oriented images into
an ordered array and merge them into a
three-dimensional reconstruction-in essence,
creating a virtual crystal.
The power of single-particle image analyis as a
tool for structural genomics was recently
demonstrated in a project led by Nogales. Working
with proteins prepared in the lab of UC Berkeley
professor Robert Tjian, Nogales and Frank Andel
used the combination of electron microscopy and
single particle image analysis to produce the first
3-D models of the protein machinery that initiates
the transcription of the genetic code of dna for
the subsequent production of new proteins. This
machinery is actually a complex of transcriptional
factor (tf) proteins that include TFIID, TFIIA and
After a strand of dna has been unwound and
unzipped in preparation for protein production, the
TFIID protein binds to an exposed dna section
precisely where a genetic message begins. Once
TFIID recognizes and binds to a gene on a strand of dna, it interacts with rna polymerase
so that the genetic code is transcribed into messanger rna, which then carries the
information out of the cell's nucleus and into its cytoplasm where protein assembly takes
"Our model gives us a good idea as to how TFIID works in concert with TFIIA and TFIIB
to initiate and regulate the transcription of protein coding genes," says Nogales. "We
show a horseshoe-shaped structure surrounding a central cavity inside of which
recognition and binding to dna takes place."
Structural determinations for some domains and other components of the TFIID, TFIIA
and TFIIB proteins have been determined through x-ray crystallography but the size of
TFIID, coupled with the difficulties posed in trying to crystallize all of the tf proteins
together, has so far precluded the use of x-ray diffraction for imaging the entire complex.
Consequently, until now the overall shape and relative position of the components within
the complex were a mystery.
TFIID is a horseshoe-shaped protein that starts the process of gene transcription by binding
to an exposed strand of DNA precisely where a genetic message begins. The central cavity of
the horseshow is sized to easily fasten around a single strand of DNA.
Because Nogales and her colleagues did not have to crystallize the tf complex, they
could work with a relatively small amount of sample and still produce an image of the
entire transcriptional machine. In less than seven months, they had their 3-D
reconstruction at a resolution of 35 angstroms.
"Our study shows that electron microscopy is a good technique for studying biological
complexes of proteins and nucleic acids that are too large or too fragile to be crystallized
for x-ray diffraction studies," says Nogales, who credits Andel for the single-particle
analysis work. "Our study also shows that single-particle methodology is a useful
technique for the structural characterization of mega-Dalton transcription complexes."
At a resolution of 35 angstroms the shapes of TFIID and its companion proteins plus their
relative positions within the tf complex can be clearly seen. When this electron
microscopy information is combined with the x-ray data on various substructures within
the complex-a technique dubbed "hybrid crystallography"-Nogales and her colleagues
expect to find further clues as to how the transcriptional machinery comes together.
(More about the Nogales lab at http://cryoem.berkeley.edu.)
Robert Glaeser, a professor of biophysics at UC Berkeley with a joint appointment in
Berkeley Lab's Life Sciences and Physical Biosciences Divisions, is a pioneer and world
authority on electron crystallography. Glaser says that hybrid crystallography is raising "a
great amount of enthusiasm" among the scientists in his field because "it is the
complexes rather than the individual components that really begin to bring structural
biology into the realm of functional genomics."
Glaeser compares x-ray crystallography with identifying all the individual components that
go into an auto engine block-spark plugs, cam shaft, valves, etc. "The structure of each
of these components is very interesting and enlightening but you also want to know how
the spark plug fits into the block, the order in which the valves open and close, and so
forth, and how all of that is accomplished," he says.
Glaeser, Nogales, and Downing have recently begun using an electron microscope at
Berkeley Lab's National Center for Electron Microscopy (ncem) that is equipped with a
field emission gun which enables it to direct highly focused electron beams at 200
kilovolts (kVs) of energy onto a sample. This sharpens the resolution and boosts the
contrast to yield better quality images than the lower-powered electron microscopes
they'd previously used. The three are overseeing the acquisition of new custom-built
electron microscope that will accelerate electron beams to 300 kV which should improve
resolution and image quality even further.
"This new microscope will allow us to get the maximum resolution possible in our images
while doing the least amount of damage to our samples," says Nogales. "With the addition
of new detector technology, we will also be able to collect our data much faster and
Between the new microscope and the facilities at ncem, the Berkeley Lab researchers
hope to be able to record up to 3,000 images a day. Still, electron microscopy and hybrid
crystallography won't match synchrotron-based x-ray crystallography for atomic-level
details and rate of throughput. Another approach to electron microscopy, however, might
Glaeser, in collaboration with Downing and Nogales, plus Ravi Malladi and Esmond Ng of
the National Energy Research Scientific Computing Center (nersc), is exploring the
viability of applying the enormous computational powers of a supercomputer to electron
microscopy imaging techniques.
With a supercomputer, rather than working with a few tens of thousands of images as is
now done, it should be possible to collect and merge as many as a million images of a
single non-crystallized protein, then reconstruct these images into a 3-D model at a
resolution comparable to that achieved with x-ray crystallography. What's more, with
supercomputers at nersc capable of making trillions of calculations every second, such
reconstructions could be done in less than half-a-day. Glaeser calls this approach
"crystallization in silico," the equivalent of using a computer to perform the difficult task
of protein crystallization.
"At present, the software that exists to do the
merging of data from a single particle was
written for workstation computers and little has
been done to take advantage of supercomputer
capabilities," he says.
Glaeser and his collaborators aim to make
crystallization in silico "an important player in
structural genomics" by looking into ways of
automating image data-collection, particle
identification, and the final merging of data from
"With further improvements in image quality,
more closely approximating what the laws of
physics will allow, the particle size that can be
handled by this technique could push down to
the range that includes all but the smallest
proteins," Glaeser says.
"At that stage, it might become the method of choice for solving the structures of any
protein that has not already been crystallized."
Zooming in on the details
With x-ray crystallography to determine the 3-D structures of protein components and
electron microscopy to show how these components fit into complexes, there is still a
need to zoom in for the finer details of electronic properties in order to see how a protein
machine performs its chemical functions. The best techniques for this work are x-ray
absorption spectroscopy (xas), and extended x-ray absorption fine structure (exafs).
Both techniques are based upon the energy spectrum produced when one or more core
electrons (those whose energy orbitals are closest to the nuclei of an atom) absorb
x-rays. In xas, the electron is bumped up to a higher energy orbital and the resulting
spectrum provides important information on how the host atom's electrons are
configured. In exafs, the electron is ejected from the atom as a photoelectron and the
resulting spectrum provides extremely accurate measurements of electron bond lengths.
"X-ray absorption spectroscopy is a wonderful complement to x-ray crystallography,"
says pbd chemist Karen McFarlane. "We can look at a specific type of atom within the
protein and see who its neighbors are, and we can look at it under several different
environments or after we've manipulated it."
McFarlane oversaw the design and construction of a new experimental endstation at als
beamline 9.3.1, a bend magnet beamline that generates x-ray photons in the 2 to 6
thousand electron volt (keV) range. These photons are ideal for the study of atoms such
as sulfur, chlorine, and calcium, which are minor but critically important protein
constituents. For example, a sulfur-containing peptide, glutathione, helps prevent
oxidative and free-radical damage to the cell, and strengthens red blood cells while
protecting white blood cells.
The new endstation at beamline 9.3.1 features a
sample chamber with a liquid helium cryostat for
cooling protein molecules down to 10 Kelvin in
order to minimize radiation damage and allow
exafs in addition to xas data collection. Pressure
within the chamber can be varied or experiments
can be done in vacuum, and samples can be
studied in a solid or liquid state. This unique
chamber was built by pbd physicist Mel Klein, who
is the principal investigator for research at the
endstation, working in collaboration with fellow
pbd chemist Vittal Yachandra.
"Until now, most xas and exafs experiments have
been done on the metal centers in metalloproteins
while experiments with sulfur, chlorine, calcium
and other lower-Z atoms have been rare," says
McFarlane. "We plan to use our experimental
station extensively for sulfur in many different
proteins, peptides, and inorganic complexes. We
also have plans for probing chlorine and calcium in
the Photosystem II protein complex."
In addition to providing electronic and structural
information that will complement x-ray
crystallography data, x-ray spectroscopy
techniques can also be used to perform
comparison measurements that will answer
questions neither technique could answer alone.
For example, such comparison measurements could help determine whether there are
differences in the active sites of proteins when they are in solution as opposed to when
they have been crystallized. Experiments on the xas/exafs endstation at beamline 9.3.1
are scheduled to begin early next summer.
Making protein movies
Whether focusing on the biggest, smallest, or finest details of protein structures, all of
the techniques so far discussed have one thing in common-all provide static images. Yet
protein machines are dynamic when they perform their functions. Just as a photograph of
a car at rest, no matter how high the image quality, yields little appreciation for what the
car will do when it moves, scientists will need to see a protein in action to fully
understand its purpose.
New technologies now being developed make it possible to obtain dynamic images of
proteins. Rather than providing composite images reconstructed from an ensemble or
aggregate of a select type of protein molecule, these new technologies enable scientists
to study and even physically manipulate a single individual molecule.
Shimon Weiss, a physicist who holds a joint appointment with the pbd and the Materials
Sciences Division (msd), is one of the recognized leaders in single-molecule
spectroscopy. He explains the advantage of characterizing and manipulating an individual
"In contrast with ensemble measurements, single-molecule experiments provide
information on distributions and time trajectories that would otherwise be hidden. In
particular, single-molecule experiments can be used to measure intermediates and follow
time-dependent pathways of chemical reactions that are difficult or impossible to
synchronize at the ensemble level."
The general strategy behind single molecule
spectroscopy is to attach dye molecules to
various sites along a protein. These dye
molecules fluoresce in a specific color when
zapped by laser light. Tracking the intensity
and location of fluorescent emissions over
time reveals any changes that may be taking
place in the protein's structure. A variation of
this strategy that is especially valuable for
measuring protein conformational changes is
"fluorescence resonance energy transfer" or
To use fret, scientists tag a protein with a
pair of dye molecules, one acting as an energy
"donor" and the other as an energy
"acceptor." The fret effect is observed when
the donor is photo-excited and its
fluorescence energy matches the energy the
acceptor will absorb. If the protein contracts
so as to bring the two tags closer together,
the fret effect is strengthened. As the protein
stretches, moving the tags further apart, the fret effect weakens.
"By performing fret measurements together with one or more of the single-molecule
manipulation techniques, it will be possible to correlate local protein structural changes
with macromolecular function," says Weiss.
One of the biggest obstacles towards achieving this goal is the limitations of current
fluorescent-dye molecules, but help is on the way. Recently Weiss and msd chemist Paul
Alivisatos announced the development of a new type of fluorescent probe made from
nanometer-sized crystals of semiconductors. These semiconductor nanocrystals offer a
distinct advantage over conventional dye molecules in that they emit multiple colors of
light, which means they can be used to label and measure several biological markers
"The use of semiconductor nanocrystals should allow us to do unique fret experiments,"
says Weiss. "For example, labeled molecules could be made to emit different colors at
different times of an event."
Alivisatos, an authority on the production by chemical means of semiconductor
nanocrystals, says, "The development of semiconductor nanocrystals for biological
labeling would give biologists an entire new class of fluorescent probes for which no small
organic molecule equivalent exists."
In an experiment on mouse tissue cells called 3T3 fibroblasts, a core nanocrystal of
cadmium selenide was enclosed within a shell of cadmium sulfide and this core-shell
combo was then enclosed within a shell of silica for water solubility and biocompatibility.
Since earlier research by Alivisatos had shown that the color of light emitted by a
semiconductor nanocrystal depends upon its size, the mouse cells were labeled with two
different sized core-shells. The silica surfaces of the core-shells were modified to
selectively control their placement within the cells. Smaller nanocrystal core-shells, which
fluoresced green, were modified to penetrate the nucleus of each cell; the larger
core-shells, which emitted red light, were modified so that they would attach themselves
to actin filaments along the outer cell membrane.
Fluorescence Resonance Energy Transfer or FRET spectroscopy features a donor and an
acceptor dye molecule attached at different points along a sample. When the donor is
photo-excited at longer distances from the acceptor, fluorescence comes exclusively from
the donor. As the space between them contracts, fluorescence is increasingly transferred
to the acceptor.
Confocal microscopy images showed that cell nuclei had been penetrated with the green
probes and the actin fibers had been stained red. The green and red labels were clearly
visible to the naked eye and could be photographed in true color with an ordinary
camera. Berkeley Lab is unique in that it is home to world-class facilities in the four main
technological areas required for solving protein structures-synchrotron-based x-ray
crystallography, nmr spectroscopy, electron microscopy, and supercomputing. Through
its close ties to the Berkeley campus of the University of California, it brings together
some of the top researchers in the biophysical sciences. The Laboratory is therefore well
positioned to help meet what has been called the grand challenge in biology,
characterizing and understanding how a cell's protein machinery works.
As pbd director Graham Fleming has said, "Nothing that takes place inside a living cell is a
random occurrence; everything is being run by those amazing protein machines."
The Department of Energy's Office of Science is the single largest supporter of basic research in the physical sciences in the United States and is working to address some of the most pressing challenges of our time.