A wide range of biological processes rely on complexes between ribonucleic acids (RNAs) and proteins. Determining the three-dimensional structures of RNA-protein complexes is crucial to elucidate the relationship between structure and biological function. X-ray crystallography represents the most widely used technique to characterize RNA-protein complexes at atomic resolution; however, determining their three-dimensional structures remains challenging. RNase contamination can ruin crystallization experiments by degrading RNA in complex with protein, leading to sample heterogeneity, and the conformational flexibility inherent in both protein and RNA can limit crystallizability. Furthermore, the three-dimensional structure can be difficult to accurately model at the typical diffraction limit of 2.5 Å resolution or lower for RNA-protein complex crystals. At this resolution, phosphates, which are electron dense, and bases, which are large, rigid, and planar, tend to be well resolved and easy to position in the electron density map, whereas other features, e.g., sugar atoms, can be difficult to accurately position. This chapter focuses on methods that can be used to overcome the unique problems faced when crystallizing RNA-protein complexes and determining their three-dimensional structures using X-ray crystallography.
Keywords: COOT; Crystallization; Electrophoretic mobility shift assay; RCrane; RNA-binding domain; RNA-binding protein; RNA–protein complex; RNA–protein interaction; X-ray crystallography.