More Staff Names Coming Soon
Much of the lab’s activity focuses on the structure
and function of tubulin, the main protein in microtubules.
Microtubules play a vital role in the life of all eukaryotic
cells, as they are involved in organelle movement, separation
of chromosomes during cell division, maintenance of cell
shape and other critical cellular activities.
The assembly and disassembly of microtubules at particular
times are essential steps in the cell cycle. These processes
are closely regulated, and interference with the regulatory
mechanisms can lead to cell death. These properties have
made tubulin both a fascinating specimen for biophysical
studies and a useful target for anti-cancer drugs. It is
important to understand how tubulin molecules interact with
each other as well as with a large number of other proteins
and ligands in these activities in order to have a full
understanding of the life of the cell. As a first step in
this direction we have determined the structure of tubulin
by electron crystallography. In further work we are extending
our understanding of the structure and learning more about
the processes that give tubulin its unique properties. We
study the interaction of tubulin with drugs that stabilize
microtubules and the interactions with some of the proteins
that regulate the microtubule cytoskeleton. Through studies
of tubulin in complexes with ligands such as aluminum fluoride
and pentalysine, and using genetic manipulation, we are
investigating factors that give microtubules their particular
metastable character. This work is aimed at developing a
rational understanding of the functional mechanisms of microtubule
dynamics and may reveal the underlying mechanism of microtubule
stabilization, eventually allowing development of new, more
effective drugs targeted to tubulin.
We are also developing instrumentation and technology to
improve data collection in electron microscopy, especially
applied to protein structure determination. One major project
is the development of a new charge-coupled device (CCD)
camera for intermediate voltage electron microscopes (IVEMs).
CCDs have found wide application in certain aspects of electron
microscopy. They provide much faster availability of image
and diffraction data than photographic film, and this improvement
in turnaround time has allowed a tremendous increase in
the efficiency of specimen evaluation, microscope operation
and automation, and data recording. However, the CCD performance
is seriously compromised when the microscope is operated
much above 100 kV, due to the decrease in efficiency of
the scintillator, which decreases the signal level, and
increased lateral scattering of the electrons within the
scintillator, which decreases the resolution. Together these
effects can produce a detective quantum efficiency (DQE)
which is far below that of film, and indeed too low for
work such as low-dose imaging of proteins. The use of IVEMs
operating at 300 - 400 kV is increasing as the many advantages
of the higher accelerating voltage become more widely recognized,
so the CCD performance on these microscopes is becoming
more of a limitation. We plan to overcome the poor performance
of CCD cameras on IVEMs by decelerating the electrons before
they reach the camera. In our design the CCD is mounted
so that it can be floated to around 200 kV. When the microscope
is operated at 300 kV we will then obtain the advantages
of the IVEM but the camera will perform as well as on a
100 kV microscope. Further improvements will be made to
the scintillator to produce even better signal and resolution
characteristics than currently available. These adaptations
will produce substantial benefits in work ranging from high-resolution
structural studies of proteins and viruses to the three
dimensional study of cell ultrastructure.
On a larger scale, we are studying the molecular architecture
of some simple but intact cells. Our goal is to apply the
same frozen-hydrated specimen preparation methodology used
for molecular studies to map out the locations of the major
protein complexes in small bacteria. Recent advances suggest
that whole microbial cells could be imaged by electron tomography
to "molecular" resolution, sufficient to locate
and identify large macromolecular complexes in the native
state within their cellular contexts. We plan to develop
and test this technique on two microbes, one chosen for
its ideal imaging characteristics, and the other for its
potential Department of Energy mission relevance. Information
gained by this technique will be critical to four stages
of the DOE Microbial Cell Project: (1) complete functional
characterization of each gene product, (2) identification
of complexes and pathways, (3) fine localization and quantification
of cell components, and (4) understanding the effects of
microbe customizations. These stages represent some of the
basic goals of all of cell biology, so that developing the
technology in the context of the MCP will have very broad
implications for the study of other organisms.
Senior Staff Scientist/
Life Sciences Division
One Cyclotron Rd.
Berkeley, CA 94720
E. Nogales, S. G. Wolf, and K. H. Downing Structure of the
tubulin dimer by electron crystallography. Nature 391, 199-203
E. Nogales, K. H. Downing, L. A. Amos and J. Lowe Tubulin
and FtsZ form a distinct family of GTPases. Nature Struct.
Biol. 5, 451-458 (1998).
E. Nogales, M. Whittaker, R. A. Milligan and K. H. Downing
High-resolution model of the microtubule. Cell 96, 79-88 (1999).
J. Löwe, H. Li, K. H. Downing and E. Nogales Refined
structure of ab-tubulin at 3.5 Å. J. Mol. Biol. 313,
N. V. Hud and K. H. Downing Cryoelectron microscopy of l
phage DNA condensates in vitreous ice: The fine Structure
of DNA toroids. Proc. Natl. Acad. Sci. USA 98, 14925-14930
S. Zhong, V. M. Dadarlat, T. Head-Gordon, R. M. Glaeser and
K. H. Downing Modeling chemical bonding effects for protein
electron crystallography: The transferable fragmental electrostatic
potential (TFESP) method. Acta Cryst. A 58, 162-170 (2002).
H. Li, D. J. DeRosier, W. V. Nicholson, E. Nogales and K.
H. Downing Microtubule structure at 8 Å resolution.
Structure 10, 1317–1328 (2002).