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X-ray analysis is best known as a technique for solving the three-dimensional structures of molecules ranging from small chemical entities to highly complex macromolecular assemblies. Today, however, X-ray structure determination also plays a key role in biomedical research for unraveling the binding mode of low molecular weight ligands to their protein targets, and high-throughput crystallography is deployed as a screening technology for the identification of novel ligands and the development of innovative drugs. In this chapter we reflect on how X-ray analysis can answer the three main questions about protein ligands: who binds, where and why? The intricacies of ligand binding energetics, and how these can be illuminated by structural information, are discussed in depth and illustrated with many concrete, informative examples. A short introduction to the physical principles of the technique is also provided, while the practical aspects, technical limitations and potential synergies with other biophysical techniques are examined in more detail. The chapter is intented for researchers and students in the chemical, pharmacological, biophysical and biological sciences who rely on crystallographic data for their own work or require an insight into how their research could benefit from X-ray analysis. Up to date references pointing to authoritative reviews and research articles are provided for those who wish to delve deeper into the subject.

The use of biophysical methods for characterizing ligand binding to macromolecules of biological interest is presented in terms of three questions: Who? Where? Why? The “who” question addresses the problem of identification of the ligands binding to a given target, the “where” question addresses the problem of determining the site of binding, and the “why” questions try to establish the physical (especially thermodynamic) basis of binding. This chapter establishes this framework, which will be followed in the rest of the book.