Deriving RNA topological structure from SAXS

Structures of well-folded RNA molecules can be determined with atomic resolution by either X-ray crystallography, cryo-EM, or NMR spectroscopy, but those of conformationally-flexible RNAs often are difficult to study with these methods. However, flexible RNAs have biological relevance and likely represent the majority of the RNA conformational space. Due to the high electron density of the phosphate-sugar backbone, RNA is very sensitive to small-angle X-ray scattering (SAXS), and SAXS data can be recorded with sub-μM concentrations and under near-physiological solution conditions without the need for labeling. For these reasons, SAXS has significant advantages over other techniques for obtaining global structural information of flexible RNAs in the form of molecular envelopes or low-resolution topological structural models. The SAXS-derived information is extremely valuable for bridging secondary structure data, often determined by other techniques, with a three-dimensional structure description. In this chapter, we present a detailed account of the principle, algorithms, and experimental and computational protocols for topological structure determination of RNA molecules in solution. To illustrate the applications of the methodology, we provide several case studies that cover a broad spectrum of the RNA conformational landscape.