Time‐resolved X‐ray Crystallography


Time‐resolved crystallography is a collection of methods whereby biological reactions proceed in crystals of a macromolecular catalyst, and structures of intermediates formed on the reaction pathway are determined using X‐ray crystallography. Such experiments allow investigators to visualize directly the mechanism of enzyme‐catalysed reactions or other biological processes.

Keywords: laue diffraction; trapping; enzyme mechanism; photocage; crystallography

Figure 1.

Polychromatic Laue X‐ray diffraction pattern. The pattern shown is a computational representation of the diffracted X‐rays produced from a crystal of isocitrate dehydrogenase using an incident X‐ray beam bandpass of 0.95–1.55 Å and a maximum resolution for observed spots of 2.4 Å. For this crystal, which belongs to space group P43212, a = b = 105.1 Å, c = 150 Å, more than 50% of the unique diffracted X‐rays are collected from a single exposure. Typical exposure time to collect a diffraction pattern is 1 ms.

Figure 2.

General methods of intermediate accumulation for time‐resolved crystallographic studies. (a) A rapid initiation event, usually a photoreaction, induces the formation of a substrate complex ‘A’ with a rate constant of kstart. The reaction proceeds and encounters a rate‐limited intermediate ‘I’ which decays with a rate constant kcat. The lifetime of the intermediate during this initial reaction event is dictated by kcat. The occupancy or concentration of I throughout the crystal during this time period is dictated by the initial concentration of A produced by the photoreaction, and by the ratio of kstart to kcat. (b) A high occupancy, rate‐limited, steady‐state complex accumulates throughout the crystal as a result of diffusion of substrate into the crystal solvent channels. The occupancy or concentration of the rate‐limited intermediate I is a function of the rate of diffusion relative to kcat, as is the time required to achieve steady state. In principle, the intermediate I can be maintained indefinitely; in practice, the crystal will often display a limited period of stability under multi‐turnover conditions.



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Further Reading

Altarelli M, Schlachter F and Cross J (1998) Making ultrabright X‐rays. Scientific American 279(6): 66–73.

Cruickshank DWJ, Helliwell JR and Johnson LN (eds) (1992) Time‐resolved Macromolecular Crystallography. New York: Oxford University Press.

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Stoddard, Barry L(Mar 2002) Time‐resolved X‐ray Crystallography. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0003046]