Analysis of riboswitch structure and ligand binding using small-angle X-ray scattering (SAXS)

Small-angle X-ray scattering (SAXS) is a powerful tool for examining the global conformation of riboswitches in solution, and how this is modulated by binding of divalent cations and small molecule ligands. SAXS experiments, which typically require only minutes per sample, directly yield two quantities describing the size and shape of the RNA: the radius of gyration (Rg) and the maximum linear dimension (Dmax). Examination of these quantities can reveal if a riboswitch undergoes cation-induced compaction. Comparison of the Rg and Dmax values between samples containing different concentrations of ligand reveals the overall structural response of the riboswitch to ligand. The Kratky plot (a graphical representation that emphasizes the higher-resolution SAXS data) and the P(r) plot or pair-probability distribution (an indirect Fourier transform, or power spectrum of the data) can provide additional evidence of riboswitch conformational changes. Simulation methods have been developed for generating three-dimensional reconstructions consistent with the one-dimensional SAXS data. These low-resolution molecular envelopes can aid in deciphering the relative helical arrangement within the RNA.