Small-angle X-ray scattering (SAXS) is a low-resolution method for the structural characterization of biological macromolecules in solution. Information about the overall structural features provided by SAXS is highly complementary to X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryo-electron microscopy, which are high-resolution methods. SAXS not only provides the shape, oligomeric state, and quaternary structure of folded proteins and protein complexes but also allows for quantitative analysis of flexible biomolecules. In this chapter, the most relevant SAXS procedures for structural characterization of flexible macromolecules, including intrinsically disordered proteins (IDPs), are presented. The sample requirements for SAXS experiments on protein solutions and the sequence of steps in data collection and processing are described. The use of the advanced data analysis tools to quantitatively characterize flexible proteins is presented in detail. Typical experimental issues and potential problems encountered during SAXS data measurements and analyses are discussed.
Keywords: Flexible macromolecules; Functional complexes; Intrinsically disordered proteins; Macromolecular structure; Small-angle X-ray scattering (SAXS); Solution scattering.