The overall objective of the proposed research is a major enhancement in methods for macromolecular structure analysis. Recent advances demonstrate that anomalous scattering can be used for straightforward determination of three-dimensional structure from a single protein crystal. Realization of the full potential of the new methods requires further development in theory, computation, biochemistry, and diffraction. These developments will be pursued in the context of analyzing particular crystal structures of biological significance. The overall objective is embodied in four specific aims: (1) We propose to continue our efforts to develop the theoretical basis and computational implementation of methods for exploiting the effects of anomalous scattering. Our main emphasis will be on multiwavelength anomalous diffraction (MAD) studies. (2) We propose to optimize the experimental procedures for conducting MAD experiments. For the most part these experiments will be conducted at the Hughes Synchrotron Resource that we are developing at Brookhaven National Laboratory. (3) We propose to study various means for introducing suitable scattering centers into macromolecules. An emphasis will continue on perfecting bacterial systems for expressing recombinant proteins that quantitatively incorporate selenomethionine. (4) We propose to perform the methodology developments in the course of applications to macromolecular crystal structures. Specific plans are described for studies on several systems of considerable biological and medical significance.
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