Macromolecular Structure Determination by X‐ray Crystallography

Abstract

Abstract For more than 50 years, single‐crystal X‐ray diffraction has remained at the forefront of structural studies of biological macromolecules and complex molecular machines. Diffraction occurs when X‐rays interact with the electrons in the crystallised macromolecule or a complex. Importantly, only amplitudes, not phases, are recorded during an X‐ray diffraction experiment. This ‘phase’ problem is resolved by incorporating electron‐dense, anomalous scatterers into the protein or nucleic acid molecules. Major technical advances in crystal handling, synchrotron beamlines, free‐electron lasers, detector technology, software development and computer hardware have allowed X‐ray crystallography to become essentially a routine method for obtaining high‐resolution images of biomolecules. Much of our insights into macromolecular structure/function relationships are, in fact, based on single‐crystal X‐ray crystallography. Key Concepts: X‐ray crystallography is one of the fundamental methods of structural biology and remains the most widely used technique to study the structure/function relationships of macromolecules. X‐ray diffraction is not an imaging technique as the scattered X‐ray intensities need to be associated with phases to assemble an image. The phase problem in X‐ray crystallography can be overcome by determining the anomalous scattering generated by suitable heavy atoms. Crystallisation of biological macromolecules remains a bottleneck in X‐ray crystallography.