Time resolved macromolecular crystallography

Reviews of this topic have been given by Bartunik (1983), Moffat (1989a), Moffat and Helliwell (1989) and Hajdu and Johnson (1990). 

Unfortunately, there are difficulties which have severely limited the method to a very few examples. The cryosolvents used often modify the kinetic behaviour of the molecule of interest. In addition, crystallinity is often lost. 

A time resolved crystallographic experiment has three key components reaction initiation, reaction monitoring and X-ray data acquisition and analysis. The X-ray aspects of such experiments are feasible now. However, the real challenge lies in devising suitable ways of reaction initiation and monitoring in the crystal. Progress to date will now be covered. Table 10.8 provides details of examples of time resolved crystallography. 

The single crystal sample must obviously be of a size and quality suitable for high resolution X-ray diffraction. Hence, a structural reaction must be initiated uniformly and promptly through the whole of the crystal. Also, in such a process the crystal must not be damaged. It is essential that spatial uniformity be achieved in less time than the lifetime of all subsequent molecular intermediates whose imaging is required. 

A reaction can be initiated chemically, e.g. by altering the concentration of a reactant or physically by changing the temperature or pressure. The simplest method of reaction initiation is to allow a reactant to diffuse into a crystal mounted in a flow cell (Wyckoff et al 1967) (see also figure 5.32). However, diffusion is slow taking many seconds for a crystal of a few hundred microns. Diffusion is only appropriate for initiating a relatively slow reaction. However, it has been used in the monochromatic SR study of glycogen phosphorylase b as detailed in the case study below. 

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Moffat, K. 1989. Time resolved macromolecular crystallography. Annu. Rev. Biophys. Biophys. Chem. 18 309-23. 

D. W. J. Cruickshank, J. R. Helliwell and L. N. Johnson eds., Time-Resolved Macromolecular Crystallography , Oxford University Press, 1992, (ISBN 0-19-855781-7). 

Moffat, K. 2003. The frontiers of time resolved macromolecular crystallography VIovjcs and chirped -ray pulses. Faraday Discuss. 122 65-88. 

Moffat, K. A. (1989). Time-resolved macromolecular crystallography. Annual Review of Biophysics and Biophysical Chemistry, 18, 309-32. 

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