Chapter 20
Studying RNA–Protein Complexes Using X-Ray
Crystallography
Andrew P. Turnbull and Xiaoqiu Wu
Abstract
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 threedimensional structure can be difficult to accurately model at the typical diffraction limit of 2.5 A ˚ 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.
Key words X-ray crystallography, Crystallization, RNA-binding protein, RNA-binding domain,
RNA–protein complex, RNA–protein interaction, Electrophoretic mobility shift assay, RCrane,
COOT
1 Introduction
Many biologically important ribonucleic acids (RNAs) carry out
their cellular functions in complexes with RNA-binding proteins
(RBPs). RBPs account for between 5 and 10% of the eukaryotic
proteome and play fundamental roles in many biological processes
such as RNA metabolism, translation, DNA damage repair, and
gene regulation. RBPs can bind many different types of RNAs, e.g.,
transfer RNA (tRNA), ribosomal RNA (rRNA), and messenger
RNA (mRNA), with diverse structures ranging from singlestranded RNAs through to complex three-dimensional motifs and
typically engage RNA through one or more RNA-binding domains
(RBDs). In addition, a recent proteome-wide analysis has identified
Tina Daviter et al. (eds.), Protein-Ligand Interactions: Methods and Applications, Methods in Molecular Biology, vol. 2263,
https://doi.org/10.1007/978-1-0716-1197-5_20, © Springer Science+Business Media, LLC, part of Springer Nature 2021
423
Studying RNA–Protein Complexes Using X-Ray
Crystallography
Andrew P. Turnbull and Xiaoqiu Wu
Abstract
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 threedimensional structure can be difficult to accurately model at the typical diffraction limit of 2.5 A ˚ 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.
Key words X-ray crystallography, Crystallization, RNA-binding protein, RNA-binding domain,
RNA–protein complex, RNA–protein interaction, Electrophoretic mobility shift assay, RCrane,
COOT
1 Introduction
Many biologically important ribonucleic acids (RNAs) carry out
their cellular functions in complexes with RNA-binding proteins
(RBPs). RBPs account for between 5 and 10% of the eukaryotic
proteome and play fundamental roles in many biological processes
such as RNA metabolism, translation, DNA damage repair, and
gene regulation. RBPs can bind many different types of RNAs, e.g.,
transfer RNA (tRNA), ribosomal RNA (rRNA), and messenger
RNA (mRNA), with diverse structures ranging from singlestranded RNAs through to complex three-dimensional motifs and
typically engage RNA through one or more RNA-binding domains
(RBDs). In addition, a recent proteome-wide analysis has identified
Tina Daviter et al. (eds.), Protein-Ligand Interactions: Methods and Applications, Methods in Molecular Biology, vol. 2263,
https://doi.org/10.1007/978-1-0716-1197-5_20, © Springer Science+Business Media, LLC, part of Springer Nature 2021
423