X-ray crystallography at subatomic resolution

he diffraction ofX-rays bymolecular crystals is the technique of reference for obtaining three-dimensional information about atomicpositions and interactions, information essential for the comprehension of the function and the molecular mecha­ nisms. In the case ofsmall molecules, very precise high resolution measurements allowed the observation ofhydrogen atoms ofand bond electronic densities. Thus, relations could be established between the deviations from standard stereochemistry ofspheri­ cal atomic models and the chemical reactivity. In the case of biological macromolecules, one could correlate the spatial arrangement of the components of proteins and nucleic acids to their biological function. These two types of studies progressed independently during the two last decades, primarily because ofthe limited resolution of the macromolecular crystallographic results, 2 to 3 A in the majority of the cases, against 0.5 A or better for the small mole­ cules. The resolution, which is the minimal separation of the crystal plans giving place to an observable X-ray diffraction spot, is indeed an essential parameter of a crystallographic study. It is directly related to the minimum distance separating the details of the electronic density. A resolution of 2 A is sufficient to distin­ guish peptides from a protein or the bases of a nucleic acid, but not the individual atoms, and even less the bond densities. In the last ten years, various technical improvements, ranging from better techniques ofexpression and crystallisation to the use ofsynchrotron sources for measurements ofdiffraction and algo­ rithms of multipolar and quantum modelling, made it possible to improve considerably the resolution and the quality of the macromolecular models 1. Biological structural studies with resolutions between 1.5 and 0.9 A became more current. In this range of resolution, the individual atoms can be clearly distin~ guished and the hydrogen atoms start to appear. As the errors of atomic position are reduced of an order of magnitude (typically from 0.2 to 0.03 A), the variations observed from standard stere­ ochemistrystart to be significant. Since 1997, several structures were solved with a resolution bet­ ter than 0.9 A,in particular crambin 2, subtilisin 3 and aldose reductase 4. With such a resolution, the level of the details observed in the best ordered areas approaches that of the small molecules studies. The hydrogen atoms and the bond densities are dearly visible, and the atomic errors ofco-ordinates are reduced another order of magnitude (-0.003 A), which makes the stereo­ chemical differences highly significant. Estimation of the atomic charg~s starts to be possible. In whatfollows, we will discuss two ofthese cases, crambin and aldose reductase, from the crystallisation and the determination of the structure to the relations between structural details and the reactivity.