ABSTRACT
Introduction In order to fully understand biological processes, a knowledge of the atomic structure of the macromolecules involved is essential. e most common method of structure determination is X-ray crystallography, and the many protein and DNA structures already determined have provided incredible insights into the molecular underpinnings of life. Most X-ray crystal structures, however, are static average snapshots of a molecule and only yield information about a single state of a complex reaction. In addition, the deleterious eects of radiation damage can cast uncertainty on the validity of the model itself. More information about function can be obtained via kinetic crystallography, and one form this can take is soaking experiments. ese take advantage of the fact that, as long as the active site is not blocked by crystal packing, and turnover does not require any major structural rearrangements that could disrupt the crystalline lattice, many enzymes retain their catalytic activity in the crystalline state. By soaking in a substrate and ash-cooling the crystal in liquid nitrogen, intermediate species can be freezetrapped for structure determination (Kovaleva & Lipscomb, 2007; Schlichting & Chu, 2000; Katona et al., 2007). Identication of the trapped species, however, is limited by the resolution of the diraction data obtained and the occupancy of the trapped state. is is often insu cient to unambiguously assign the chemical intermediate observed in the electron density map, especially in cases in which there are no large conformational changes in the ligand and/or protein. is ambiguity highlights the need for spectroscopic methods complementary to X-ray crystallography in structural biology to identify the exact chemical state of the trapped species.
