Three-Dimensional Macromolecular 
and Cellular Structure 
David A. Agard, Ph.D. — Associate Investigator 
Dr. Agard is also Associate Professor of Biochemistry and Biophysics at the University of California, San 
Francisco. He did his undergraduate work at Yale University with Frederic Richards, Hal Wyckoff, and 
Thomas Steitz. He received his Ph.D. degree in chemical biology from the California Institute of 
Technology, where he studied with Robert Stroud and began a continuing collaboration with John Sedat. 
His postdoctoral work was done on high-resolution electron microscopic crystallography at the MRC 
Laboratory of Molecular Biology in Cambridge, England, with Richard Henderson. There he also began 
the cloning of the a-lytic protease gene with Sydney Brenner. 
WE study chromosome structure in close col- 
laboration with John Sedat (HHMI, Univer- 
sity of California, San Francisco); hence only a 
subset of these studies will be discussed here. 
Our primary aim in this area is to provide a physi- 
cal basis for understanding chromosome behav- 
ior and function by directly determining the 
three-dimensional structure of chromosomes as a 
function of both transcriptional state and the cell 
cycle stage. Current efforts are aimed at deter- 
mining how fibers of nucleosomes are folded into 
higher-order structures within the chromosome 
and what role specific chromosomal proteins 
play in determining these structures. 
We are using intermediate voltage electron mi- 
croscope (IVEM) tomography to examine higher- 
order chromosome structure. In the past year sig- 
nificant improvements in the quality of the 
three-dimensional reconstructions have led to 
the first new insights into the structure of the 30- 
nm fiber (in collaboration with Chris Woodcock, 
University of Massachusetts) . We are now begin- 
ning to be able to trace the paths of the 30-nm 
chromatin fibers within telophase chromosomes. 
It is clear that the existing models of chromatin 
structure are seriously flawed. In addition, we 
have made dramatic steps toward our goal of fully 
automating the complex task of collecting three- 
dimensional IVEM tomography data. This will 
simplify the arduous task of collecting 100-150 
images and should dramatically reduce radiation 
exposure (from 3 hours to about 5 minutes) . Sig- 
nificant progress has also been made on under- 
standing the mechanism of image formation for 
thick specimens and on applying a powerful new 
approach to the problem of electron microscopic 
(EM) reconstruction. Together with a new stain- 
ing approach and cryopreparation methods, 
these improvements should allow us to trace the 
paths of the 30-nm fibers throughout the chro- 
mosome and will finally make this exciting struc- 
tural approach available to the general cell 
biologist. 
Structural Basis of Enzyme Specificity 
By combining solution kinetic analysis, x-ray 
crystallographic structural analysis, and site- 
directed mutagenesis, we have been able to 
probe the structural basis for substrate specificity 
in unprecedented detail, using a-lytic protease as 
a model system. The availability of peptide bor- 
onic acid inhibitors, which act as excellent 
mimics of the reaction transition state, or nearby 
intermediates, has allowed us to use x-ray crystal- 
lography to examine the complex set of interac- 
tions between enzyme and substrate that mediate 
specificity. 
By mutation, we have been able to alter dramat- 
ically the pattern of substrate specificity while 
maintaining or even increasing enzyme activity. 
Approximately 40 high-resolution, extremely 
well-refined crystal structures have now been de- 
termined and analyzed. Detailed structural analy- 
ses of three mutants as free enzymes and as com- 
plexes have provided surprising insights into the 
mechanism of specificity and have indicated the 
crucial role that protein flexibility plays in selec- 
tivity. During the past year we have made numer- 
ous other mutations and examined their kinetic 
and structural propenies. Of particular note is a 
mutation that alters specificity indirectly by 
changing active-site flexibility. This remarkable 
finding is the first demonstration that residues 
beyond those that directly contact the substrate 
can play a significant role in determining the pat- 
terns of specificity. 
Not long ago we began a collaboration with 
Vladimir Basus (University of California, San Fran- 
cisco) to perform a complete analysis of a-lytic 
protease structure in solution by nuclear mag- 
netic resonance (NMR) methods. We have 
now made '^N,''C doubly labeled enzyme and 
have collected sufficient high-resolution three- 
dimensional NMR data sets to permit complete 
backbone assignment — a significant task for such 
a large protein. Currently we are working on the 
assignments and on the collection of data for the 
complete side chain assignments. We anticipate 
that the NMR data will provide insights into 
correlated motions within the enzyme and be 
crucial for the folding studies described below. 
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