Introduction 
tional role of biologically interesting molecules 
can best come from understanding the forms of 
the molecules themselves. As Francis Crick, the 
co-discoverer of the double-helical structure of 
DNA (see Figure 9), remarked: "to understand 
function it is essential to study structure." It was 
with this in mind that in 1985 the Institute made 
a substantial commitment to develop a new Pro- 
gram in Structural Biology. 
At present, x-ray crystallography is the most 
powerful approach for visualizing the three-di- 
mensional structures of large molecules (com- 
monly called tnacromolecules) . An essential pre- 
requisite for x-ray analysis is the availability of 
crystals of the molecule or molecular complex 
that are suitable for recording the diffraction of 
x-rays. The production of crystals, in turn, re- 
quires chemically homogeneous preparations. 
Moreover, molecules that are not spatially uni- 
form (too "floppy") must be broken down or 
molecularly dissected into defined and rigid com- 
ponents. For example, to crystallize antibody mol- 
ecules, it was important first to cleave them into 
their principal fragments, Fab and Fc, because 
these pieces are normally connected by a flexible 
hinge. And because of antibody diversity (de- 
scribed in the section on immunology) , it became 
necessary to study Fab fragments from monoclonal 
immunoglobulins. A continuing challenge to 
structural biologists is the development of strate- 
gies for crystallizing membrane proteins — by sol- 
ubilizing them with detergents, dissecting them 
into pieces, or altering them by mutation. 
Genetic engineering has transformed struc- 
tural biology. This approach, which makes it pos- 
sible to produce large quantities of pure proteins, 
also allows an investigator either to choose a suit- 
able fragment for study or to modify genetically 
the molecule to be crystallized. Other method- 
ological advances in crystallography itself are 
transforming the field by extending the range of 
problems that can be tackled routinely. 
There are essentially four stages in determining 
a structure by x-ray diffraction analysis (Figures 
25 and 26): 1) diffraction experiments (data col- 
lection); 2) complex computations that pro- 
duce, in effect, an image of the molecule(s) in 
the crystal; 3) interpretation of the computed 
image in terms of a molecular model; and 4) re- 
finement of the model by further computation. 
Synchrotron x-ray sources, which are a thou- 
sand or more times stronger than conventional 
laboratory x-ray generators, are making it possi- 
ble to study structures that could not previously 
be solved. (HHMI is currently developing a 
synchrotron resource for use by the biological 
community at the National Synchrotron Light 
Source at Brookhaven National Laboratory on 
Long Island.) Recent examples from HHMI labo- 
ratories are the human class I major histocompati- 
bility antigen and the DNA virus SV40. At the 
same time, jxjsition-sensitive x-ray detectors have 
greatly extended the applications of conven- 
tional radiation sources. Lastly, novel computa- 
tional methods have made the production of a 
molecular image (phase determination) less 
dependent on extensive ancillary data from 
heavy-atom modified crystals and have made re- 
finement of models less cumbersome and more 
objective. 
In the 1950s and 1960s, x-ray crystallography 
revealed the structures of the first biologically 
important molecules, including DNA, hemoglo- 
bin, and insulin. In the 1970s it revolutionized 
the field of enzymology by making it possible to 
visualize directly the active sites of enzymes. In 
the 1 980s it made comparably far-reaching con- 
tributions to virology, immunology, and mem- 
brane biology by revealing the structures of vi- 
ruses, antibodies, and a photosynthetic reaction 
center. What can we expect in the 1990s? It 
seems reasonable to predict the following: 
1. Structures of different classes of proteins 
or protein/nucleic- acid complexes. Three de- 
cades of biological crystallography have left sev- 
eral major areas unexplored. For example, we 
have yet to know what any of the major proteins 
of the cytoskeleton and of cellular motility look 
like (actin, myosin, tubulin, and so forth). We 
have yet to visualize any of the membrane recep- 
tors referred to above, and we have yet to see an 
ion channel, a ribosome, an RNA polymerase, or a 
ribozyme; and, with the exception of transfer 
RNAs (tRNAs), little is known of the three-dimen- 
sional structure of most RNAs and RNA-protein 
complexes. Progress toward some of these goals 
is reported in this volume; others will no doubt 
be achieved before long, as more and more 
workers are drawn into the field and as new tech- 
niques are developed. 
2. Time-resolved images of events at the ac- 
tive site of an enzyme. New ways of using 
synchrotron x-ray radiation permit very rapid 
measurements of diffraction data, so that in prin- 
ciple it should be possible to follow the struc- 
tural changes that occur during an enzymatic re- 
action. If we understood these changes, it might 
be possible to develop enzymes with usefully al- 
tered properties and to synthesize enzyme inhibi- 
tors with enhanced specificity. 
Ivii 
