Core 2 - Structural Cell Biology (SCB) Core PROJECT SUMMARY/ABSTRACT A major challenge for cancer structural biology in SBDR4 is to structurally define the DNA repair (DR) proteins and assemblies that maintain genomic integrity. However. DR proteins and complexes have encoded flexibility and function in spatially and temporally coordinated complexes that make their structural characterizations so challenging. With SBDR investigators, Core B, the Structural Cell Biology (SCB) Core overcomes these extreme challenges by combining macromolecular crystallography (MX) and small angle X-ray scattering (SAXS) to structurally characterize almost every DR target. SAXS measures flexibility, conformations, and assemblies, while MX provides atomic-level structural information. Together, SAXS and MX are synergistic and complementary: they reveal the impact of sequence variations, the means to dissect multiple functions, and the mechanisms whereby flexible and dynamic DR machinery functions. With the SCB Core based at the Berkeley Advanced Light Source Synchrotron, the Core will uniquely provide SBDR laboratories unrivaled access 1) to beamline facilities for MX and small angle X-ray scattering (SAXS) data collection, 2) to expertise of beamline scientists, 3) to the latest developments in data analysis, and 4) to development of novel methods for their Projects, as needed. SBDR members collect their own data or have data collected and analyzed for them. The SCB Core will train and assist researchers, design and perform experiments. This will enable all Projects, regardless of structural expertise, to optimally and efficiently address Project structural and mechanistic Aims in ways that typical laboratories and beamline visits cannot or do not do. The impact of the SCB Core staff's close collaborations and expertise in MX and SAXS are evident from their many publications with SBDR members in all Projects. SCB Core-Project interactions will provide knowledge of DR structures, conformations, and mechanisms as follows:
Aim 1 is to provide SAXS measurement of flexibility, conformations, and assemblies in solution, with added capabilities of high throughput SAXS and size exclusion chromatography in-line with SAXS data collection.
Aim 2 is provide atomic MX structures of components and complexes, with added services for SAXS screening for crystallizability and with new MX hardware developments.
Aim 3 is to further develop hybrid methods for combining SAXS and MX, as well as observing protein and DNA conformations using time-resolved SAXS. SCB Core collaborations address cross-Project hypotheses that 1) DR proteins adopt specific conformational and/or assembly states to regulate binding, activation and product release; 2) distinct functional conformations provide mechanisms to dissect, predict, and control activities with small-molecules and mutations, and 3) controlling conformations, product release and pathway handoffs provide the means to control synthetic lethality independently of active sites. With our SAXS metrics and with planned innovations in MX and SAXS, the SCB Core will obtain usable SAXS and/or MX data for every SBDR project in collaborations with the EMB Core and most SBDR members.
Core 2 - Structural Cell Biology (SCB) Core PROJECT NARRATIVE Like a picture is worth a thousand words, structures are worth a thousand experiments. Structures provide highly informative clues to how proteins work and in cases of patient mutations, clues as to how they fail. The SCB Core will collaborate with SBDR members to maximally obtain structural information on their DNA repair targets.
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