Publisher Summary This chapter discusses phase determination from multiwavelength anomalous diffraction (MAD) measurements. The MAD approach to macromolecular structure determination has potential advantages for accuracy and convenience in phase evaluation. Isomorphism is intrinsically perfect; an algebraically exact analysis is possible; relative scattering strength and phasing power increase with scattering angle; and all required diffraction data can be measured from a single crystal. The virtual immortalization of crystals through freezing makes these advantages real and practical. Although many of the procedures in MAD phasing are in common with crystallographic practice, there are also a number of practical steps unique to this methodology. The chapter discusses the basic foundations of the method, presents the design and execution of experiments, and describes the steps and alternatives for data analysis.

[28] Phase determination from multiwavelength anomalous diffraction measurements.

The X-ray diffraction data collection system, with a Weissenberg camera, an imaging plate, and image reader BA100, using SR was constructed in the Photon Factory for protein crystallography. It allows us to collect a full data set with one crystal in 2.5 h by rotation of a sample around a single axis. The high accuracy and extended resolution of the collected data were confirmed by comparison with the data obtained from other detectors and results from the structure determination. Further improvements in the function and accuracy of this system are discussed.

X-ray diffraction data collection system for modern protein crystallography with a Weissenberg camera and an imaging plate using synchrotron radiation

In conventional x-ray diffraction experiments on single crystals, essentially monochromatic x-rays are used. If polychromatic x-rays derived from a synchrotron radiation spectrum are used, they generate a Laue diffraction pattern. Laue patterns from single crystals of macromolecules can be obtained in less-than 1 second, and significant radiation damage does not occur over the course of an exposure. Integrated intensities are obtained without rotation of the crystal, and individual structure factors may be extracted for most reflections. The Laue technique thus offers advantages for the recording of diffraction patterns from short-lived structural intermediates; that is, for time-resolved crystallography.

https://science.sciencemag.org/content/sci/223/4643/1423.full.pdf

X-ray Laue Diffraction from Protein Crystals

This review describes the utilisation of synchrotron X-radiation (SR) in protein crystallography. For the general reader a brief discussion is given of the basics of protein structure analysis including an outline of the problems of sample radiation damage, the inherent weakness of individual reflections and the large amounts of data that need to be collected as well as the 'phase problem' of crystallography. The properties of synchrotron radiation emitted by relativistic electrons in simple circular orbits in bending magnets and by more sophisticated electron motions induced in wiggler or undulator magnets are discussed. The impact of newer high brightness synchrotron radiation sources in protein crystallography is assessed. The instrumentation required to monochromatise and focus the radiation onto typically small protein crystal samples and to detect the diffraction pattern is described. Both step scanning and energy dispersive techniques to optimise anomalous dispersion are covered. A gazetteer is given of the instruments providing data collection facilities for protein crystallography which are available on SR sources around the world. The modifications that are needed to standard data processing techniques are dealt with, taking account, for instance, of the position, angle and wavelength correlatable properties of photons incident to a sample from an SR source; properties which affect reflection prediction in both camera or diffractometer work.

http://iopscience.iop.org/article/10.1088/0034-4885/47/11/001/pdf

Synchrotron X-radiation protein crystallography: instrumentation, methods and applications

Macromolecular X-ray crystallography underpins the vigorous field of structural molecular biology having yielded many protein, nucleic acid and virus structures in fine detail. The understanding of the recognition by these macromolecules, as receptors, of their cognate ligands involves the detailed study of the structural chemistry of their molecular interactions. Also these structural details underpin the rational design of novel inhibitors in modern drug discovery in the pharmaceutical industry. Moreover, from such structures the functional details can be inferred, such as the biological chemistry of enzyme reactivity. There is then a vast number and range of types of biological macromolecules that potentially could be studied. The completion of the protein primary sequencing of the yeast genome, and the human genome sequencing project comprising some 105 proteins that is underway, raises expectations for equivalent three dimensional structural databases.

/pdf/trends-and-challenges-in-experimental-macromolecular-16zssq2l9j.pdf

Trends and challenges in experimental macromolecular crystallography.

In principle, it is possible to phase the X-ray reflections from a macromolecular crystal which contains one heavy atom per molecule, without having recourse to the conventional method of multiple isomorphous replacement, by making use of anomalous dispersion effects alone1. To do this, it is necessary to measure each reflection over a narrow range of wavelengths centred on an absorption edge of the heavy atom, where f′ and f″, the real and imaginary anomalous components of the scattering factor, are varying rapidly. The availability of powerful synchrotron radiation sources with a continuous energy spectrum has stimulated interest in such phasing methods, but previously experiments1 have been limited to making measurements on only one reflection at a time and/or to measuring at only a few discrete wavelengths near the absorption edge. We are developing a method at the Daresbury Synchrotron Radiation Source (SRS) which produces an energy profile along an axis of each and every diffraction spot in a screenless oscillation photograph. We show here that the approach is feasible using a single crystal o heptahydrido bis (diisopropylphenyl)phosphine rhenium using the LIII absorption edge of rhenium at a wavelength of 1.1772 A.

Optimized anomalous dispersion in crystallography: a synchrotron X-ray polychromatic simultaneous profile method

A new instrument is being established on the SRS wiggler beamline at Daresbury. The design consists of a toroid focussing mirror to produce a point focussed white beam of X radiation of an intensity sufficient for millisecond and sub-millisecond Laue exposures from protein crystals. The wavelength band available for focussed Laue experiments will be variable by means of a plane mirror with an adjustable graze angle and by adjustable thickness beryllium filters. Unfocussed white beam will also be available to provide the full spectral range of the wiggler source. New, larger film cassettes and changing mechanisms are being designed to use the available data more efficiently (by decreasing spatial overlap and increasing the number of recording ‘singlet’ reflections). A double crystal monochromator option will be available for rapidly tunable monochromatic data collection. Design principles based on theoretical work and ray-tracing are dealt with in the paper.

A new protein crystallography station on the SRS wiggler beamline for very rapid Laue and rapidly tunable monochromatic experiments: I. Design principles, ray tracing and heat calculations

This report describes the use of the MAXRAY raytracing package [1,2] in assessing the design of a focussing mirror for the protein crystallography (PX) station due to be constructed on the storage ring “SIBERIA−2” in Moscow. Focussing optics are required for PX because of the large discrepancy in the size of the diverging synchrotron radiation beam and the protein crystal sample (typically 0.3 × 0.3 × 0.3 mm3). The efficiency of X-ray collection is investigated as a function of grazing angle, magnification and misalignment error for both toroidal and ellipsiodal mirror forms, and these results are used to recommend a flexible optical arrangement.

A.W. Thompson

Papers

Optimized anomalous dispersion in crystallography: a synchrotron X-ray polychromatic simultaneous profile method

A new protein crystallography station on the SRS wiggler beamline for very rapid Laue and rapidly tunable monochromatic experiments: I. Design principles, ray tracing and heat calculations

Report on the raytracing of a focussing mirror for a PX station on the storage ring “SIBERIA-2”