OTHER DESIGNATIONS: Protein crystallography (PX).

PURPOSE: PX is the most powerful method for the determination of the three-dimensional structure of large biological molecules (macromolecules). Areas addressed include:

• Therapeutic drug design
• Enzyme mechanisms
• Supramolecular structure
• Molecular recognition
• Nucleic acids
• Structural genomics
• High-throughput crystallography

HOW THE TECHNIQUE WORKS: X-rays are passed through crystals of the macromolecule under study. The x-rays are scattered by the atoms of the crystal, producing a diffraction image that gives information on the structure of the crystals. In multiwavelength anomalous diffraction (MAD), x-rays of different wavelengths are used on the same crystal to detect small variations in the diffraction intensity at different energies due to the presence of a heavy atom. This provides information about the relative phases of the diffracted beams, crucial for reconstructing an image of the molecule and without which additional measurements must be made or some prior structural knowledge exploited.

UNIQUENESS: Because macromolecules are large and flexible, their crystals tend to be small, imperfect, and weakly diffracting. In many cases, the intensity, small beam size, and collimation of a synchrotron beam is vital for successful results. The MAD phasing method also requires tunability of wavelengths. Because MAD uses only a single crystal and can provide phases very rapidly, it is a popular technique among crystallographers today, and beamlines optimized for these experiments are among the most oversubscribed.

EXAMPLES:

A Membrane Protein Structure Worthy of a Nobel Prize
Anthrax Toxin — Working Toward an Antidote
Enzyme Structure Helps Unravel Mysteries of DNA
The Many Faces of Botulinum Neurotoxin
Molecular Movies — The Time-Resolved Structure of Myoglobin

Virtually all communication between a cell and its environment is mediated by membrane proteins. They are critical in a variety of biological functions, including photosynthesis, vision, neural transmission, pathogenesis, and drug resistance. Membrane proteins control the electrochemical potentials that generate nerve impulses, transduce the signaling functions of hormones, and even generate adenosine triphosphate (ATP) — the cell’s source of energy. Even though they represent approximately 30% of proteins coded by genomes, they are dramatically underrepresented in the Protein Data Bank. They are notoriously difficult to crystallize. Synchrotron x-ray sources are essential for making advances in this field. Work on the voltage-dependent potassium channel, awarded the 2003 Nobel Prize for chemistry, is a perfect example of the dramatic impact that structural studies of membrane proteins have in the understanding of cellular function. Certain membrane proteins open and close to regulate ion conduction in response to changes in cell-membrane voltage. These “life transistors” help to control electrical activity in muscles and nerves. The structure, showing 4 red-tipped “paddles” that open and close in response to positive and negative charges, answers the question of how this kind of channel functions as a voltage-dependent switch, driving muscle and nerve activity in all living organisms.

Y. Jiang, A. Lee, J. Chen, V. Ruta, M. Cadene, B.T. Chait, and R. MacKinnon, “X-ray structure of a voltage-dependent K+ channel,” Nature 423, 33 (2003).

Anthrax makes a deadly cocktail of three toxin proteins that flood the bloodstream, leading to rapid death if the infection is not treated in its early stages. Even antibiotic treatments can fail when the anthrax bacterium, Bacillus anthracis, has already produced lethal levels of toxins. The poisonous protein called lethal factor (LF) rapidly blocks signals to recruit immune cells to fight the infection. Another enzyme, edema factor (EF), causes the release of fluid into the lungs and is deadly on its own. Protective antigen (PA) facilitates the entry of these toxin proteins across the cell membrane, and into target cells, through its complex pore-forming channel. LF is the greatest source of damage in highly fatal cases of inhalation anthrax. An antitoxin that stops LF would be a vital addition to combined therapy with existing treatments (antibiotics, anti-PA antibodies, critical care). Scientists have taken a big step forward in developing a drug to inhibit the LF toxin. Small molecules were screened to identify chemical compounds that can block LF. Crystals of LF bound to these candidate inhibitors were made and x-ray crystallography was used to analyze the interactions of these compounds with LF. The research concluded that the most effective inhibitors targeted the active center via hydrophobic interactions and also deprived LF of zinc.

R.G. Panchal, A.R. Hermone, T.L. Nguyen, T.Y. Wong, R. Schwarzenbacher, J. Schmidt, D. Lane, C. McGrath, B.E. Turk, J. Burnett, M.J. Aman, S. Little, E.A. Sausville, D.W. Zaharevitz, L.C. Cantley, R.C. Liddington, R. Gussio, and S. Bavari, “Identification of small molecule inhibitors of anthrax lethal factor,” Nature Structural & Molecular Biology 11, 67 (2004).

Before a cell can begin to divide or differentiate, the genetic information within the cell’s DNA must be copied, or “transcribed,” onto complementary strands of RNA. RNA polymerase II (pol II) is an enzyme that, by itself, can unwind the DNA double helix, synthesize RNA, and proofread the result. When combined with other molecules that regulate and control the transcription process, pol II is the key to successful interpretation of an organism’s genetic code. However, the size, complexity, scarcity, and fragility of pol II complexes have made analysis of these macromolecules by x-ray crystallography a formidable challenge. A team of structural biologists has met this challenge using data obtained from synchrotron facilities. The resultant high-resolution model of a 10-subunit pol II complex suggests roles for each of the subunits and will allow researchers to begin unraveling the intricacies of DNA transcription and its role in gene expression.

P. Cramer, D.A. Bushnell, J. Fu, A.L. Gnatt, B. Maier-Davis, N.E. Thompson, R.R. Burgess, A.M. Edwards, P.R. David, and R.D. Kornberg, “Architecture of RNA polymerase II and implications for the transcription mechanism,” Science 288, 640 (2000).

Botox® face lifts and botulism disease are both based on a neurotoxin from the bacterium Clostridium botulinum. The toxin, often described as the most lethal substance known, is a member of the clostridal neurotoxins (CNTs) group, which block muscle contractions. When injected into a person’s face, the effect is a lessening of wrinkles. When ingested, the toxin paralyzes muscles, including those of the internal organs, causing sickness and death. The toxin is also used in medicine for conditions such as uncontrolled blinking, lazy eye, and involuntary muscle contractions. Nerve cells cause muscles to move by delivering the neurotransmitter acetylcholine into muscle cells. CNTs paralyze muscles by blocking acetylcholine delivery. CNTs enter nerve cells then find and cut SNARE proteins, the machinery responsible for acetylcholine delivery. The inactivation of nerve cells lasts for three to six months. Scientists have solved the first crystal structure of a CNT bound to a SNARE. They found extensive contact between the toxin and its target. In kinetic experiments based on the crystal structure, the authors found that the toxin wraps the target SNARE protein around itself. This ensures both target specificity and proper positioning for cutting the SNARE. Knowing the structure of the CNT–SNARE interaction furthers understanding of the toxin’s mechanism and may lead to drugs that can treat CNT poisoning.

M.A. Breidenbach and A.T. Brunger, “Substrate recognition strategy for botulinum neurotoxin serotype A,” Nature 432, 925 (2004).

Time-resolved crystal- lography enables the capture of myoglobin “movie” sequences.

Crystallographers are no longer confined to static observations of protein structures. The availability of extremely intense, multiwavelength, pulsed synchrotron x-ray sources has reduced exposure times enough to capture “movie sequences” of fundamental molecular processes. This approach has been applied to studies of myoglobin, the iron-based molecule responsible for oxygen transport in muscles. Absorption of a photon by myoglobin breaks a bond between the central iron atom and a carbon monoxide molecule, initiating a series of spectroscopic and structural changes, ultimately followed by rebinding of the carbon monoxide. The entire photolysis, relaxation, and rebinding processes occur in less than 5 milliseconds at room temperature. To observe this, the carbon monoxide was photodissociated by a 7.5-nanosecond laser pulse, and the subsequent structural changes were probed by 150-picosecond or 1-femotosecond x-ray pulses at delay times ranging from 1 nanosecond to 1.9 milliseconds. Researchers are now extending this approach to several other light-sensitive signaling systems that are chemically and biologically diverse and are developing new techniques that will enhance the time resolution from the nanosecond range, first to a few hundred picoseconds and perhaps ultimately to femtoseconds.

V. Srajer, Z. Ren, T.-Y. Teng, M. Schmidt, T. Ursby, D. Bourgeois, C. Pradervand, W. Schildkamp, M. Wulff, and K. Moffat, “Protein conformational relaxation and ligand migration in myoglobin: A nanosecond to millisecond molecular movie from time-resolved Laue x-ray diffraction,” Biochemistry 40, 13802 (2001). Link: moffat.bsd.uchicago.edu.