Electron density maps anomalous dispersion methods

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. 

The most demanding element of macromolecular crystallography (except, perhaps, for dealing with macromolecules that resist crystallization) is the so-called phase problem, that of determining the phase angle ahkl for each reflection. In the remainder of this chapter, I will discuss some of the common methods for overcoming this obstacle. These include the heavy-atom method (also called isomorphous replacement), anomalous scattering (also called anomalous dispersion), and molecular replacement. Each of these techniques yield only estimates of phases, which must be improved before an interpretable electron-density map can be obtained. In addition, these techniques usually yield estimates for a limited number of the phases, so phase determination must be extended to include as many reflections as possible. In Chapter 7,1 will discuss methods of phase improvement and phase extension, which ultimately result in accurate phases and an interpretable electron-density map. 

If the electron density map was originally phased using isomorphous replacement or anomalous dispersion methods, then a number of statistical criteria are usually available,... 

The a vaiues are a measure of the electron-density variation in the protein and solvent regions, and the ratio of these numbers is a measure of the contrast between the two regions. Since anomalous dispersion data were used to phase the maps, the map for the correct hand will show greater contrast. In this case, the original direct-methods sites give rise to greater contrast thereby indicating that these sites do correspond to the correct enantiomorph. 

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