Anomalous dispersion phase determination using

X-ray crystallography is a widely used teclmique for the three-dimensional structure analysis of RNA molecules. The extraction of information from X-ray diffraction patterns from RNA crystals requires proper phase determinations using methods such as multiwavelength anomalous dispersion in which placement of specific heavy atoms is required. Seleiuum-modi-fled ribonucleotides have been used for these purposes by Du et al. (36), because the selenium atom does not seem... 

Variable wavelength approaches to phase determination using anomalous dispersion were discussed in the late 1950s by Okaya and Pepinsky as well as Mitchell, in the 1960s by Herzenberg and Lau and Karle and pioneered by Hoppe and Jakubowski. Technically these methods and others have only become really feasible with the synchrotron. 

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 anomalous components of the total scattering are wavelength dependent and the use of radiation close to an absorption edge may increase or optimise the contribution due to the anomalously scattering atoms. Ramaseshan (1962) pointed out that data collected at multiple wavelengths optimising the anomalous dispersion effects would improve the quality of phase determination. 

The use of anomalous dispersion has led to many results of interest in chemistry and biochemistry such as the steric course of certain chemical reactions.The uses for detailed studies of chirality and for protein relative phase determination will now be discussed. 

Anomalous dispersion can also be used as an aid in the determination of phase angles. It was realized early on that anomalous scattering from the heavy atom in a derivative could be used to resolve the phase ambiguities if a single heavy-atom derivative is all that is available. 

Anomalous dispersion measurements have proved to be very useful as an aid in phase determination in protein structure determination. 

Because part of the anomalous dispersion component is jt/2 out of phase with the isomorphous, real component, the net observable effect is a breakdown of Friedel s law regarding the perfect equality of the magnitudes of and If-h-k-i- That is, the two need not be absolutely equivalent but can demonstrate some slight difference A I anom = fhki — f-h-k-i- This difference will normally be imperceptible and within the expected statistical error of most X-ray diffraction intensity measurements, but with care in data collection, and judicious choice of X-ray wavelength, it can be measured and used to obtain phase information in conjunction with isomorphous replacement phase determination, or even independently, as described in Chapter 8. 

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. 

In the past 15 years a number of technical advances have made it possible to maximize and precisely measure anomalous dispersion differences, and more powerful mathematical approaches have been devised to optimize its use for phase determination. The experimental problem was always to amplify the difference between and and obtain its... 

This method of isomorphous replacement, together with anomalous dispersion data collection is, to date, the principal method that has been successful for phase determination of macromolecules. It was first used successfully for proteins, myoglobin byjohn Kendrew and coworkers (14), and hemoglobin by Max Perutz and co-workers (15). Unfortunately, it is common to find that, although a heavy-atom solution has been soaked into a protein crystal, no regular (ordered) substitution has occurred, and solutions of other heavy-atom compounds must be tried. 

Small-angle X-ray scattering (SAXS) experiments using synchrotron radiation (SR) are performed at present mainly in the areas of real time scattering and anomalous dispersion.1 Typical applications are the study of melting or recrystallisation of semicrystalline polymers [4, 5], phase separation of alloys [6], muscle diffraction and stopped flow experiments on dissolved biopolymers [7, 8]. Anomalous dispersion has been exploited in order to determine partial structure factors in alloys [9] or polymers containing heavy atoms [10],... 

Ramaseshan (1964) discussed the possibilities of optimising the anomalous dispersion with different X-ray tubes. Hoppe and Jakubowski (1975) used Ni and CoKa radiation to collect two data sets about the iron edge in erythrocruorin in this pioneering study, phases were determined with a figure of merit of 64% (mean phase error—50°). 

---