John A. Tainer
Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

This lecture will address both theoretical and practical concepts, concerns and considerations for the effective combination of SAS and crystallography to define accurate macromolecular structures in solution where multiple domains, conformational changes and complexes can result in dynamic structures. Crystallography supplies unparalleled structural detail for mechanistic analyses; however, it is restricted to describing low energy conformations of macromolecules within crystal lattices. SAS offers complementary information about macromolecular folding, unfolding, aggregation, extended conformations, flexibly linked domains, shape, conformation, and assembly state in solution, albeit at the lower resolution range of about 50 to 10 A resolution, but without the size limitations inherent in NMR and electron microscopy studies. We designed and built the SIBYLS synchrotron beamline at the Advanced Light Source to interconvert between a SAXS and a crystallography endstation quickly (under an hour). One of our goals for this beamline is to encourage the use of these combined techniques, so examples from data collected at SIBYLS will be presented. Detailed aspects of SAS experiments will be considered with a focus on strategy and tactics for the application of data analysis and interpretation tools for macromolecular structures. For example, SAS results can be key to determining correct molecular mechanisms for complexes involving functionally-important conformational changes (Yamagata and Tainer, 2007). Similarly, SAS can identify conformations that reflect changes from crystal packing (Tsutakawa et al., 2007). We conceptually divide the SAXS experiment into four major steps: data collection, data evaluation, data analyses, and solution structure modeling (Putnam et al., 2007). Each of these steps will be considered for exemplary systems. The methods discussed will provide the basis to examine molecular interactions in solution and to characterize macromolecular flexibility and conformational changes that have become increasing relevant for accurate understanding, simulation, and prediction of mechanisms in structural cell biology and nanotechnology.

Putnam, C.D., Hammel, M., Hura, G. L., and Tainer, J. A. (2007) X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution. Quarterly Reviews in Biophysics, 40, 191-285.

Tsutakawa, SE, Hura, GL, Frankel, KA, Cooper, PK, and Tainer, JA (2007) Structural analysis of flexible proteins in solution by small angle X-ray scattering combined with crystallography. J. Structural Biology, 158, 214-223.

Yamagata, A, and Tainer, JA (2007) Hexameric structures of the archaeal secretion ATPase GspE and implications for a universal secretion mechanism. The EMBO Journal, 26, 878-890.

Date/time: Friday, 24 October, 14:00