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Scientific Opportunities: Life Sciences

Overview | Chronology | Macromolecular Crystallography | Structure & Dynamics | Biological & Biomedical Imaging

Macromolecular Crystallography

Overview
X-ray crystallography has transformed our understanding of biological processes. It was X-ray diffraction that provided the first clues to the structure of the DNA double helix 50 years ago, giving profound insights into how DNA is replicated. The reality that knowledge of biological structure imparts deep insights into the mechanism of action of molecules and assemblies, and the increasing difficulty in determining those structures as they get larger, has been one of the major driving forces in the continuing development of synchrotron radiation facilities world wide.

Molecular complexes and assemblies are the challenge of the 21st century. Conventional macromolecular crystallography has benefited from nearly every improvement in synchrotron sources. The possibilities offered by increased brightness have driven researchers to attempt increasingly difficult scientific problems, especially structures of large, asymmetric assemblies, which is where the cutting-edge of structural biology currently lies.

The routine use of synchrotron radiation for single crystal diffraction studies has revolutionized macromolecular structural biology. With the availability of brighter X-ray sources, the size and complexity of macromolecules that can be studied has increased by an order of magnitude, or three orders of magnitude in mass (see figure). However, crystals of the most complex structures that are suitable for diffraction are often scarce and difficult to obtain. Therefore, continuing advances in synchrotron radiation sources, detectors, and software are required to tackle the most challenging problems, which are the ones most likely to make a significant impact on our knowledge of the functioning of living systems.

The growing use of synchrotron sources for macromolecular crystallography has increased the pressure on existing facilities to upgrade existing, or construct new, sources and beamlines. This problem is particularly acute in the Northeastern United States, where aging synchrotron sources at Brookhaven National Laboratory and Cornell University find it increasingly difficult to meet the experimental demands of a large group of crystallographers working in this region.

Biological and biomedical research has entered a new era, with an increasing emphasis on understanding the functional and physical connections between macromolecules - how the molecular components of cells and tissues are connected in biochemical pathways, cellular responses, and functioning organs. This requires first determining the structures of these components, and the field of structural biology is now poised to make invaluable contributions to our understanding of assemblies of interacting macromolecules.

NSLS-II is needed to meet the needs of scientists who will accomplish this difficult work.

Views of the structure of the T. therm ophilius 70S ribosome from the back of the 30S subunit. Structures like the 70S ribosome have relied both on synchrotron studies and cryo-electron microscopy results.

Large Molecular Assemblies
Several past successes in molecular structure determination of large complexes and assemblies are indicative of the critical role of high brilliance, high flux synchrotron sources in these studies. Early applications were the structure determination of virus particles, consisting of 180 or more proteins. Recent successes with the determination of the structure of the core of much larger viruses have shown the enormous potential of modern X-ray crystallography to reveal vast amounts of information. These and application of crystallography to large asymmetric complexes, such as ribosome complexes, were critically dependent on routine and frequent access to synchrotron radiation facilities.

Other large assemblies comprised of proteins and nucleic acids are continuously being identified and will be the target of high-resolution crystallographic studies in the future. A high brilliance X-ray source will be essential to make progress with these challenging projects.

Membrane Proteins
Membrane proteins are ubiquitous and essential to all living cells. They are involved in every aspect of cellular function, such as energy production and nerve signal transmission, and are targets within the pharmaceutical industry for the development of new therapeutics. However, only a few dozen structures of such molecules are known - a very small fraction of the total.
 

The structure of the voltage-dependent potassium (K+) ion channel with the voltage sensor paddles (in red) moving across the lipid membrane. Frequent access to synchrotron sources was essential to the insight gained to the function of the K+ channel.

The work on the voltage-dependent potassium channel (see figure), just awarded the 2003 Nobel Prize in Chemistry to NSLS user Roderick MacKinnon, is an example of the dramatic impact that structural studies of membrane proteins have in the understanding of cellular function. Voltage-dependent cation channels open and allow ion conduction in response to changes in cell membrane voltage, controlling electrical activity in nerves and muscle.

Local access to an extremely bright source of X-rays was critical to the success of MacKinnon's work, and future work in this area depends strongly on frequent access to a bright X-ray source to provide the constant feedback between the synchrotron and the biochemistry lab that is essential.

Structural Genomics
The availability of complete genome sequences for many organisms stimulates the imagination of all biologists. Since proteins are central to almost all aspects of biology and disease, Structural Genomics will have an impact on the way biological problems are addressed.

The Protein Structure Initiative, a structural genomics program funded by the National Institutes of Health - National Institute of General Medical Sciences (NIH-NIGMS), aims to provide structural information for all proteins in all naturally occurring protein sequences using a combination of experiment and comparative protein structure modeling. NSLS-II will play a major role during and after the production phase of the Protein-Structure Initiative. NSLS-II undulator beam lines dedicated to structural biology can be used to handle this load: crystals will be screened for quality; if quality is adequate, sufficient data to solve the structure will be measured and all results recorded in an experiment-tracking database.

The structure of the HIV-1 protease, with a model of Viracept, a potent, orally bioavailable inhibitor, in the active site. This structure was published by workers at Agouron Pharmaceuticals.

Drug Design
In the last 25 years, x-ray crystallography has helped target such major diseases as HIV infection (see figure), high blood pressure, and diabetes, but has not yet realized its full potential as part of the drug-design process.

Today virtually every large pharmaceutical company has a crystallography group. Recognizing the need for rapid and frequent access to synchrotron radiation, twelve of these companies operate their own beamline at the Advanced Photon Source at Argonne National Laboratory.

A critical challenge faced by pharmaceutical crystallographers is that the macromolecules studied are very often human proteins, since the aim is to treat human diseases. Human proteins can be very difficult to work with, and growing crystals large enough to study can be daunting. Another challenge is time. After the first structure is determined, there are several more steps to take before one can determine the properties that might be required of a drug. Synchrotron-based crystallography, performed at convenient, efficient synchrotron facilities like NSLS-II, helps to optimize crystallization methods and solve new structures easily.

Last Modified: May 2, 2014
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