689 Synchrotron radiation applications to macromolecular crystallography Keith Moffat * and Zhong Ren ² Progress has been rapid in the development and application of four different types of macromolecular crystallographic experiment at synchrotron hard X-ray sources: multiwavelength anomalous diffraction; studies of crystals with very large unit cell dimensions; structure determination at atomic or near-atomic resolution; and time-resolved studies. The results illustrate the interplay between the advanced technical capabilities available at new beamlines and more challenging scientific issues. Addresses Department of Biochemistry and Molecular Biology, and Consortium for Advanced Radiation Sources, The University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA * e-mail: moffat@cars.uchicago.edu ² e-mail: renz@cars.uchicago.edu Current Opinion in Structural Biology 1997, 7:689–696 http://biomednet.com/elecref/0959440X00700689  Current Biology Ltd ISSN 0959-440X Abbreviations APS Advanced Photon Source CHESS Cornell High Energy Synchrotron Source DHFR dihydrofolate reductase ESRF European Synchrotron Radiation Facility HRV human rhinovirus MAD multiwavelength anomalous diffraction MIR multiple isomorphous replacement NSLS National Synchrotron Light Source PF Photon Factory PYP photoactive yellow protein TBP TATA box binding protein TF transcription factor Introduction The past five years since the publication of Helliwell’s magnum opus [1] on the applications of synchrotron radiation to macromolecular crystallography have seen a maturation of certain applications, the successful develop- ment of new applications, and proposals for revolutionary new applications [2 • ,3]. These advances have been made largely possible by the commissioning of purpose-built insertion device beamlines at third generation sources such as the European Synchrotron Radiation Facility (ESRF), in Grenoble, France, the Advanced Photon Source (APS) at Argonne National Laboratory, USA, and (shortly) by SPring-8 outside Osaka, Japan. New beamlines have been complemented by the continuous upgrading and wider availability of beamlines at existing synchrotron facilities, such as the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory, USA, the Cornell High Energy Synchrotron Source (CHESS) at Ithaca, New York, USA, the Photon Factory (PF) at Tsukuba, Japan, and the Deutsche Elektronensynchrotron at Hamburg, Germany [4]. As the science drives the technology, the advances have been fuelled, first, by the evident success of the multiwavelength anomalous diffraction (MAD) phasing technique [5] and its consequent, wide adoption; second, by increasing numbers of experiments and refinement at atomic resolution (< 1.2 A ˚ [6,7]); third, by continued struc- ture determination of virus and multiprotein complexes that crystallize with very large unit cells — in excess of 500 A ˚ and even 1000 A ˚ (e.g. [8,9]); and fourth, by the development and successful application of time-resolved experiments on the timescales of kiloseconds, tens of seconds, milliseconds and nanoseconds [10,11 •• ,12 • ,13 •• ]. A characteristic of the field is the interplay between the science to be explored and the technology (in the broadest sense) with which to conduct it. The X-ray source, the beamline optics, the crystal manipulation devices, the detector and the software [14] are integral parts of the experiment and, for the best results, their properties have to be considered from the outset. The ability to conduct first-rate synchrotron experiments is often at least as much due to effective background reduction as to enhancement of the diffraction signal. Measurement of the highest resolution shells of monochromatic oscillation data, or of the intrinsically weak Laue diffraction patterns obtained with exceedingly brief, single X-ray pulses [15 •• ], depends critically on the reduction of the background noise. Thus, seemingly mundane experimental details, such as the beamline slit system, the air path around the crystal, the method of mounting the crystal, and the intrinsic noise and mode of operation of the detector, become important. It is ironic that, while huge sums of money are available for enhancing the signal (via source brilliance), much smaller sums are typically available for reducing the noise. The overall result has been a rapid and very substantial increase in the percentage of papers in macromolecular crystallography that utilize synchrotron radiation. Five or seven years ago, only the more adventurous crystallogra- phers used synchrotron radiation on their most challenging problems; in 1996, Hendrickson and Br¨ and ´ en [16] noted that more than 60% of the X-ray studies reported in the journal Structure used synchrotron radiation, a percentage that is matched or even slightly higher in Science, Nature and Cell. The use of synchrotron radiation is fast becoming routine for a wide variety of structural projects, while challenging new applications that are currently by no means routine continue to be developed. One point of our brief review is to illustrate why and how advances have occurred. We describe a variety