Single-wavelength anomalous dispersion technique

Excellent and detailed treatments of the use of anomalous dispersion data in the deduction of phase information can be found elsewhere (Smith et al., 2001), and no attempt will be made to duplicate them here. The methodology and underlying principles are not unlike those for conventional isomorphous replacement based on heavy atom substitution. Here, however, the anomalous scatterers may be an integral part of the macromolecule sulfurs (or selenium atoms incorporated in place of sulfurs), the iron in heme groups, Ca++, Zn++, and so on. Anomalous scatterers can also be incorporated by diffusion into the crystals or by chemical means. With anomalous dispersion techniques, however, all data necessary for phase determination are collected from a single crystal (but at different wavelengths) hence non-isomorphism is less of a problem. 

The tunability of synchrotron radiation allows for data collection at or near the x-ray absorption edge of anomalous scatterers present in the protein or crystal to provide experimental phase information. Using techniques such as multi-wavelength anomalous dispersion (MAD) and single-wavelength anomalous diffraction (SAD) researchers are now able to solve macromolecular structures in a matter of days or weeks, a process that required months, or even years, a decade ago. 

Crystals from selenium-methionine- and/ or selenium-cysteine-labeled proteins can be studied by multi-wavelength anomalous dispersion (MAD) phasing techniques that can facilitate the solution of an X-ray crystal structure from a single crystal form [11]. However, if in vivo expression systems are used to prepare selenium-labeled proteins, amino acid metabolism and the toxicity of Se-methionine can result in low protein yields and low incorporation rates. 

X-Ray diffraction from single crystals is the most direct and powerful experimental tool available to determine molecular structures and intermolecular interactions at atomic resolution. Monochromatic CuKa radiation of wavelength (X) 1.5418 A is commonly used to collect the X-ray intensities diffracted by the electrons in the crystal. The structure amplitudes, whose squares are the intensities of the reflections, coupled with their appropriate phases, are the basic ingredients to locate atomic positions. Because phases cannot be experimentally recorded, the phase problem has to be resolved by one of the well-known techniques the heavy-atom method, the direct method, anomalous dispersion, and isomorphous replacement.1 Once approximate phases of some strong reflections are obtained, the electron-density maps computed by Fourier summation, which requires both amplitudes and phases, lead to a partial solution of the crystal structure. Phases based on this initial structure can be used to include previously omitted reflections so that in a couple of trials, the entire structure is traced at a high resolution. Difference Fourier maps at this stage are helpful to locate ions and solvent molecules. Subsequent refinement of the crystal structure by well-known least-squares methods ensures reliable atomic coordinates and thermal parameters. 

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