International
Tables for Crystallography Volume C Mathematical, physical and chemical tables Edited by E. Prince © International Union of Crystallography 2006 |
International Tables for Crystallography (2006). Vol. C. ch. 3.4, pp. 166-167
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Useful recent reviews on protein crystallography at low temperatures have been written by Hope (1990) and Watenpaugh (1991
).
Crystals of biological macromolecules are very susceptible to radiation damage, and this can severely limit the amount and quality of diffraction data that can be collected per crystal. There have been relatively few systematic studies of this phenomenon (Young, Dewan, Nave & Tilton, 1993; Gonzalez & Nave, 1994
; Nave, 1995
), but one of the first effects of radiation damage is the deterioration of the high-resolution regions of the pattern, followed by increasing loss of crystallinity. Improvement of crystal lifetime in X-ray beams has been obtained by the addition of free-radical scavengers (Zaloga & Sarma, 1974
) and the replacement of the mother liquor with solutions containing 10–20% polyethylene glycol 4000 or 20000 (Cascio, Williams & McPherson, 1984
). The use of synchrotron radiation has also led to improved data-per-crystal ratios (Lindley, 1988
). The high intensity allows fast collection of data, and the high collimation permits different sections of the same crystal to be used for data collection. This is particularly useful for prismatic crystals, which can be mounted along their largest morphological axis. An alternative method of surmounting this problem, however, is to freeze the protein crystal. As the temperature is decreased, the rate of diffusion of free radicals is reduced, with a corresponding reduction in radiation damage. Appreciable reduction in diffusion rate is achieved even at 250 K, and at 100 K diffusion essentially ceases. Cryogenic measurements not only minimize radiation damage but often lead to improved resolution owing to decrease in thermal motion in the crystal. Increasing the crystal lifetime may be particularly important with respect to multiwavelength anomalous-dispersion measurements in order to derive phase information. Since crystals of biological macromolecules contain substantial amounts of solvent, typically between 35 and 80% by volume, the technical problem is to force the solvent to cool in an amorphous glass-like state, rather than as crystalline ice. The latter normally degrades the crystallinity by expansion and gives rise to powder rings, which complicate data measurement.
Cryoprotectants are normally required to avoid ice formation, and the choice of cryoprotectant will depend on the nature of the mother liquor from which the crystals have been grown. Crystals grown from high salt will usually require high salt concentration in the cryobuffer to avoid dissolution, although the addition of organic solvents may be a useful alternative. Table 3.4.1.3 lists commonly used cryoprotectants and their typical concentrations (Gamblin & Rogers, 1993
).
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The introduction of the cryoprotectant can be achieved through: (a) crystal growth in the cryoprotectant; (b) direct transfer of crystal from mother liquor into cryoprotectant buffer either in a single step or in steps of increasing cryoprotectant concentration; (c) dialysis, either direct or stepwise; or (d) exchange of liquor using a flow cell and a gradient maker.
Experience indicates that small crystals are better for cryogenic purposes, presumably because the rate of diffusion of small molecules and the rate of heat loss during rapid freezing is significantly faster than for large crystals. In most cases, there is an increase in the mosaicity (typically by a factor of 2–3), and in large specimens the increase may render the crystals useless for data collection. Successful freezing is often indicated by the crystal remaining transparent. Opacity usually indicates considerable breakdown in the crystallinity. Three commonly used methods for mounting crystals of biological macromolecules for cryogenic measurements are detailed below.
Several airstream devices have been described to cool protein crystals to around 250 K [Marsh & Petsko (1973), temperature range 253 to 303 K; Rossi (1989
), temperature range 242 to 335 K; Machin, Begg & Isaacs (1984
), 258 to 293 K; Fischer, Moras & Thierry (1985
), temperature range 263 to 293 K; Fraase Storm & Tuinstra (1986
), 250 to 350 K; Arndt & Stubbings (1987
), 248 to 353 K]. The devices of Machin, Begg & Isaacs, Fraase Storm & Tuinstra and Arndt & Stubbings involve thermoelectric modules utilizing the Peltier effect. The space available to accommodate the sample is usually very limited and care has to be taken with the length of the capillary and other aspects of crystal mounting. Hovmöller (1981
) has designed an extension to the cooling delivery tube that minimizes air turbulence at the sample. Various devices have been described that operate down to near liquid-nitrogen temperature and that can be fitted to a variety of data-collection systems. These include the rotation camera (Bartunik & Schubert, 1982
), and a universal cooling device for precession cameras, rotation cameras and diffractometers (Hajdu, McLaughlin, Helliwell, Sheldon & Thompson, 1985
). One of the more versatile devices is the cryostream described by Cosier & Glazer (1986
), which uses a pump to effectively separate the liquid-nitrogen supply from the gas outflow; this arrangement eliminates instabilities in the cooling-gas stream; the device works in the range 77.4 to 323.0 K and is commercially available (Oxford Cryosystems, England).
Cryocrystallography not only minimizes the effects of radiation damage but also often allows the collection of high-quality, high-resolution data from a single specimen. In the case of very labile systems such as ribosomal particles, it is sometimes the only means of obtaining useful diffraction data. Further, cryocrystallography permits the study of temperature effects on the structure and dynamics of biological macromolecules. In this latter regard, examples include multiple-temperature crystallographic studies on sperm whale myoglobin (Frauenfelder, Petsko & Tsernoglou, 1979; Hartmann et al., 1982
; Frauenfelder et al., 1987
) and, more recently, ribonuclease-A (Tilton, Dewan & Petsko, 1992
; Rasmussen, Stock, Ringe & Petsko, 1992
). The future will no doubt see the routine emergence of cryogenic techniques for data collection, using both conventional and synchrotron X-ray sources, from biological macromolecules, with consequent improvement in structure quality and detail.
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