Time-resolved crystallography: principles, problems and practice

Abstract

Time-resolved crystallography is founded on the belief that a complete understanding of mechanism at the molecular level demands knowledge, not just of long-lived, readily observable structures, but also of short-lived intermediates in processes such as catalysis, ligand binding and release, and protein unfolding. Synchrotron X-ray sources enable X -ray diffraction patterns of high quality to be obtained from single crystals with exposure times that are often commensurate with the lifetime of the desired intermediates. The basic tools are therefore in place. How shall they be applied effectively? Can crystallography, long regarded as a static technique, in fact encompass dynamic processes? What pitfalls remain to be overcome? A time-resolved crystallographic experiment has five main components: the X -ray source; reaction initiation; reaction monitoring by optical or other techniques; X-ray data acquisition in real time; and data reduction and analysis. These are illustrated by our recent work on photoactive yellow protein, and on the acquisition and analysis of crystallographic data using 120 ps exposures.