Modulated crystals are often encountered in crystallography. Methods for their structural characterization need further development. This research is focused on the structure determination of modulated crystals of profilin:actin. The objectives of this research are two-fold; first, to continue to develop new macromolecular X-ray crystallographic methods for the structure solution of aperiodic, modulated crystals, and second, to test the hypothetical model that the actin ''ribbon'' found in PA crystals represents a structural intermediate of filamentous actin. This research will employ protein crystal growth, X-ray diffraction data collection and structure determination. At several points during the project, innovative approaches to protein crystallography and software will be developed. The unique diffraction pattern from the modulated crystals contains unusual off-lattice satellite reflections next to the main reflections. Strategies for collecting X-ray diffraction data that detect and separate satellite reflections from the main reflections will be developed. Downstream of data collection, existing software from chemical (AKA small molecule) crystallography will be adapted and used to process the modulated diffraction pattern. From the diffraction data, the type and direction of the modulation(s) will be determined. Then additional chemical crystallography software will be extensively modified and combined with macromolecular algorithms to solve the modulated crystallographic phase problem, produce multidimensional electron density images and refine the modulated atomic structure against the experimental diffraction data. Finally, the resulting structure(s) will be analyzed within the framework of the current cell biology literature. It is anticipated that these structural results will test the validity of the ribbon-to-helix model of actin filament formation.
Broader Impacts Research will be integrated with education through partnership with the University of Nebraska-Omaha to develop undergraduate research internships and coursework in protein purification and crystallization with profilin:actin as a central theme; the formation of ''Graduate Student Training Program in Structural Biology and Biophysics'' at UNMC; supporting national conference workshops on how to solve modulated structures for the continued education of chemical and macromolecular crystallographers at all levels; and the incorporation of research results and methods into graduate-level coursework. The PI's laboratory provides a diverse and multicultural environment for the participation of underrepresented students at all levels of training in this research program.
