Abstract
Macromolecular crystallography has been highly successful in the past 60 years as it has been the predominant method to solve macromolecular structures, with more than 100,000 protein structures determined and posted to structural databases [www.rcsb.org, (Berman et al., Nucleic Acids Res 28:235–242, 2000)]. Crystallography methods are capable of determining structures at high resolution (<1.5 Å) as demonstrated by the many structures available at this or better resolution. A central objective of structural biology is not only to solve static structures but to also observe their associated dynamics to infer and explore their functions. To examine reactions that occur in biological macromolecules, time-resolved methods are required. In time-resolved crystallography, a reaction is triggered inside a crystal and the progress of this reaction is then probed by short but highly intense X-ray pulses, shorter than both the dynamics studied and the reaction trigger. Time-resolved crystallographic experiments have been successfully carried out at synchrotron X-ray sources (Moffat, Annu Rev Biophys Biophys Chem 18:309–332, 1989; Moffat, Chem Rev 101:1569–1581, 2001; Schmidt, Synchrotron Radiat News 28:25–30, 2015). Mainly cyclic reversible, and light-activated reactions were examined. Irreversible (single path) reactions, for example those catalyzed by enzymes, remain difficult to investigate. The initiation of a reaction by adding a substrate or ligand to protein crystals remains a challenge, which prevents routine applications. The arrival of X-ray free electron lasers and micro-focus synchrotron beamlines, with their intense X-ray pulses, permit the use of significantly smaller crystals. With small crystals faster diffusion times are achieved which allow for straightforward investigations of these reactions. Several successful experiments have already been reported which show how the structures of transiently occupied intermediates and their dynamics can be investigated at room temperature in real time. In this chapter we will discuss the experimental setup, feasibility, and potential impact of the new facilities on the field of enzymology.
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Kupitz, C., Schmidt, M. (2018). Towards Molecular Movies of Enzymes. In: Boutet, S., Fromme, P., Hunter, M. (eds) X-ray Free Electron Lasers. Springer, Cham. https://doi.org/10.1007/978-3-030-00551-1_12
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