14.2. MAD and MIR

Anomalous-scattering effects measured at several X-ray wavelengths can provide a direct solution to the crystallographic phase problem. For many years this was appreciated as a hypothetical possibility (Okaya & Pepinsky, 1956), but, until tunable synchrotron radiation became available, experimental investigation with the weakly diffracting crystals of biological macromolecules was limited to one heroic experiment (Hoppe & Jakubowski, 1975). Multiwavelength anomalous diffraction (MAD) became a dominant phasing method in macromolecular crystallography with the advent of reliable, brilliant synchrotron-radiation sources, the adoption of cryopreservation techniques for crystals of macromolecules, and the development of general anomalous-scatterer labels for proteins and nucleic acids. Anomalous scattering, first recognized as a source of phase information by Bijvoet (1949), has been employed since the early days of macromolecular crystallography (Blow, 1958). It has been used to locate positions of anomalous scatterers (Rossmann, 1961), to supplement phase information from isomorphous replacement (North, 1965; Matthews, 1966a) and to identify the enantiomorph of the heavy-atom partial structure in multiple isomorphous replacement (MIR) phasing (Matthews, 1966b). Anomalous scattering at a single wavelength was the sole source of phase information in the structure determination of crambin (Hendrickson & Teeter, 1981), an important precursor to development of MAD. MAD differs from these other applications in using anomalous scattering at several wavelengths for complete phase determination without approximations or simplifying assumptions.