X-ray crystallography and solution nuclear magnetic resonance spectroscopy of biological macromolecules have become important tools for understanding biological phenomena at the atomic level. The number of structures solved continues to increase rapidly due to advances in techniques such as molecular cloning, crystallization screening, data collection, and data analysis. Despite these successes, fundamental questions remain about the solution of difficult structures and about the adequate description of thermal motion and solvation of macromolecules. After data acquisition and reduction, a large variety of computational procedures are required to solve and refine a crystal or solution NMR structure. In the previous grant period we have extended the X-PLOR program by incorporating most aspects of phasing and phase improvement in X-ray crystallography. New refinement techniques were developed for both crystal and solution structures. Some of these developments were implemented by design of a high-level computer language. This high-level language allows one to easily modify existing algorithms and to even design new ones without an intervening programming step. It is proposed to pursue the following specific aims: 1. Continue the software development in order to make X-PLOR a nearly complete package for structure determination. My laboratory has recently undertaken several experimental crystallographic projects. This involvement in structure solution will enable us to test and to further develop X-PLOR by application to real problems. 2. Collect highly accurate phases for selected cases obtained by multi-wavelength anomalous diffraction and develop improved models of solvation and conformational variability. 3. Develop protocols for crystallographic refinements at low to medium resolution using all available experimental information in a reduced-variable conformational space. 4. Continue development of molecular replacement methods in the case of flexible molecules. 5 . Develop improved methods for heavy atom searches for multiple isomorphous replacement and multi-wavelength anomalous diffraction. 6. Develop improved protocols for structure determination of RNA molecules by solution NMR.
