title: Trapping reaction intermediates in macromolecular crystals for structural analyses.
abstract: The development of "time-resolved" crystallographic methods, including trapping of reaction intermediates and rapid data collection, allows the comparative study of discrete structural species formed during a macromolecular reaction, such as enzymatic catalysis, ribozyme cleavage, or a protein photocycle. The primary technical details that must be addressed in such studies are the reaction initiation, the accumulation of a specific reaction species throughout the crystal, the lifetime of that species and of the crystal under the experimental conditions, and the method used to collect X-ray data. Methods of reaction initiation range from substrate diffusion, which is appropriate for the visualization of very long-lived intermediates, to photolysis, which is appropriate for the accumulation of rate-limited species with half-lives ranging from milliseconds to nanoseconds. This review discusses various methods for initiating turnover in crystals and trapping rate-limiting species for structural studies.
authors.affiliation: Division of Basic Sciences, Program in Structural Biology, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, A3-023, Seattle, Washington 98109, USA. bstoddar@fhcrc.org
authors.forename: B L
authors.initials: BL
authors.lastname: Stoddard
mesh_terms: D001671:Biochemistry / Q000379:methods*; D002384:Catalysis; D018360:Crystallography, X-Ray / Q000379:methods*; D004583:Electrons; D004798:Enzymes / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D010782:Photolysis; D017354:Point Mutation
chemical_list: D004798:Enzymes