Core 2: Structural Cell Biology (SCB) Core

SUMMARY - CORE 2: STRUCTURAL CELL BIOLOGY (SCB) 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 2, the Structural Cell Biology (SCB) Core overcomes these extreme challenges by providing access to and expertise on cutting-edge structural biology technologies needed to probe DR proteins and complexes and not available in individual research institutions. Aim 1. Determine structures, conformations and assembly states. The SCB Core, centered at a national synchrotron facility, will provide macromolecular crystallography (MX) and small angle X-ray scattering (SAXS) data collection and beamline scientist-level expertise for SBDR members. MX provides high resolution atomic structures, while SAXS can rapidly assess flexibility, solution conformation(s), and assemblies. SCB SAXS includes ligand or buffer screening and assessing DNA bending, via GOLD-SAXS. Aim 2. Detect folds and conformations for integrative structural biology. Analysis of large complexes in the Projects, by megadalton structural mass spectrometry (MS) and with SAXS-validated structure predictions, will provide testable hypotheses on their own, or will accelerate CryoEM studies at individual institutes. Aim 3. Define molecular architectures in reconstituted systems and in the cell through synchrotron soft X-ray tomography (SXT) of reconstituted phase transitions and cells and super-resolution fluorescence measurements for quantitative measurements of DR assemblies, that would otherwise be problematic for traditional structural methods. The SCB Core is centered at the Advanced Light Source (ALS) synchrotron, where three beamlines (12.3.1, 8.3.1, and National Center for X-ray Tomography/NCXT) will provide SAXS, MX, and SXT data collection to SBDR members. For enabling access to additional structural technologies and capabilities, SCB has branches at Calgary (MS), New York (super-resolution microscopy), Oxford (MX), and Scripps (MX). All four Projects will use the SCB Core, regardless of structural expertise in member laboratories, to optimally and efficiently address Project structural and mechanistic Aims in ways that typical laboratories and beamline visits cannot or do not do. Together, these SCB modalities allow SBDR-5 interrogation of dynamic DR machines from atomic to cellular scales. The broad, long range objective of the SCB Core is to support SBDR Projects to determine the impact of sequence variations, the means to dissect multiple functions, and the mechanisms whereby flexible and dynamic DR machinery functions.