LAUSR

To determine the three-dimensional structures of proteins, we need to ‘look’ at them using electromagnetic radiation which has a similar wavelength to the interatomic distances in the molecule. These are typically around 1.5 × 10 m (= 0.15 nm =1.5 Å) but visible light has wavelengths between 400 and 700 nm which is far too long to enable us to visualise the detailed structures of proteins and viruses. We thus usually use X-rays, neutrons or electrons of much shorter wavelength: for X-rays between 0.7 and 1.7 Å which are ideal for this. However, unlike in a light microscope, we do not have any lenses that can form an image from X-rays scattering from such small objects. We have to rely on the interference patterns (so-called ‘diffraction’) formed between the X-rays as they scatter elastically (no energy loss in the sample) from crystals of the macromolecule. We then deconvolute these patterns using the tools of Mathematics (Fourier transforms) and some extra information (the ‘phases’) that we can obtain either experimentally or from a known macromolecular structure. This crystallographic method allows us to find the structures of biological molecules over an enormous range of scales from small proteins to largemacromolecular complexes and whole viruses. For instance, the structure of human insulin, a 5.8 kDa hormone vital for our metabolism of carbohydrates, fats and proteins, is ~ 20 Å in diameter, and using Xray crystallography, a structure to 1 Å resolution has been determined. At the other end of the size range, the 70S ribosome, a 2.7 MDa complex of proteins and RNA which is about 200 Å across, and the ~ 700 Å diameter and ~ 70 MDa core of the Bluetongue virus have both been solved to a resolution of 3.5 Å, allowing elucidation of biological function and mechanism. Protein crystals are typically > 30% solvent, and less than 30% of a protein surface is involved in contacts between the ordered molecules, so they can thus be thought of as very high concentration solutions (typically ~ 10 mM). Imagine many rows of synchronised swimmers lined up in a swimming pool in an ordered arrangement, with more layers of swimmers going underwater too, with each swimmer representing one protein molecule. Although overall the swimmers are dressed the same, they are not quite identical in size and demeanour. So it is with protein molecules: the structures we obtain by macromolecular crystallography (MX) are time and space averages of the many millions of molecules in the crystal, rather than being the structure of a single molecule. Thus, dynamic regions (e.g. flexible loops, the arms of the swimmers) will be less well defined in the final electron density maps than the more static parts of the protein. Functionally, it has been shown that many enzymes are active in the crystalline state, and crystal structures generally agree well with measurements from spectroscopic techniques (e.g. NMR, Fourier transform IR, fluorescence). Proteins are often observed in more than one ‘packing’ arrangement in different crystal forms, but generally, the differences in their structures are either slight (~ 0.1 Å) or conversely are potentially biologically relevant (e.g. multiple conformational states). To form a crystal, copies of a simple component (the unit cell) are packed together by translation to fill space, and this packing must be infinitely extensible in all directions. A plane lattice is an infinitely extensible construct of intersecting parallel and equidistant lines, forming identical unit lattices. In 3D, this is a basic parallelepiped (or hexagonal or trigonal sided cylinder) shaped block from which the whole volume of the crystal may be built by repetition in 3 dimensions. Its axes This article is part of a Special Issue on ‘Biophysics & Structural Biology at Synchrotrons’ edited by Trevor Sewell.