The Structural Cell Biology (SCB) Core provides critical technologies and support for the Structural Cell Biology of DNA Repair Machines (SBDR) Program. Major challenges of SBDR stem from the dynamic and coordinated assembly of large protein complexes involved in DNA repair processes. These complexes undergo functionally important conformational changes and modifications. The SCB Core will provide structural expertise and technologies suitable for SBDR project and program Aims, create a functional bridge between atomic resolution structures and molecular envelopes, and help close the gap between static crystal structures and biologically relevant, multi-component macromolecular machines. In particular, the SCB Core will provide SBDR with three distinct and complementary methods for structural analyses. (1) Multiwavelength single crystal X-ray diffraction will provide high-resolution structures of discrete states. (2) Small Angle X-ray Scattering (SAXS) will characterize the solution dynamics of protein complexes by visualizing flexible regions and induced conformational changes. (3) Scanning Force Microscopy (SFM) of single molecules, available through the Wyman lab, will provide information about DNA and protein dynamics, and will reveal structural insight into heterogeneous mixtures previously inaccessible using crystallographic or SAXS techniques. The SCB Core is designed to supply the SBDR projects with the necessary tools and proficiency to overcome the structural biology challenges inherent to analysis of large complexes. The requested funding provides staff to maximize interaction with the EMB Core and for SBDR use of the Structurally Integrated Biology for Life Sciences (SIBYLS) beamline at the Advanced Light Source (ALS) at the Lawrence Berkeley National Laboratories (LBNL). The SIBYLS beamline is a unique synchrotron resource that provides tunable wavelengths for both single crystal X-ray diffraction and SAXS. The SCB Core will develop software that addresses current limitations in the analysis of DNA repair proteins including software that will combine results from high and low resolution techniques through the systematic and objective fitting of X-ray crystal structures into molecular envelopes generated by EM and SAXS experiments. The SCB Core will test, develop, and provide advanced tools to detect and measure posttranslational modifications. Understanding the dynamic structures of macromolecular machines for DNA repair will generate insights into multi-component systems that have remained elusive through the study of individual component biomolecules. The results from the SCB Core will be applied to the understanding of cancer etiology and potential cancer diagnostics and prognostics through interactions with the UCSF Comprehensive Cancer Center.
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