Tables for
Volume F
Crystallography of biological macromolecules
Edited by M. G. Rossmann and E. Arnold

International Tables for Crystallography (2006). Vol. F, ch. 10.1, p. 197   | 1 | 2 |

Section 10.1.1. Utility of low-temperature data collection

H. Hopea*

aDepartment of Chemistry, University of California, Davis, One Shields Ave, Davis, CA 95616-5295, USA
Correspondence e-mail:

10.1.1. Utility of low-temperature data collection

| top | pdf | Prevention of radiation damage

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Since about 1985, low-temperature methods in biocrystallography have moved from stumbling experimentation to mainstay production techniques. This would not have happened without good reason. A brief discussion of the advantages of data collection at cryogenic temperatures is given.

Biocrystals near room temperature are sensitive to X-rays and generally suffer radiation damage during data measurement. Often this damage is so rapid and severe that a number of different crystals are needed for a full data set. On occasion, damage is so rapid that data collection is impossible. Crystal decay is typically accompanied by changes in reflection profiles and cell dimensions, which alter the positions of diffraction maxima, exacerbating the problem of changing diffraction intensities. The use of more than one crystal almost always introduces inaccuracies. Intensities from a crystal near the end of its usable life will have decay errors. Individual samples of biocrystals frequently have measurable differences in structure; merging of data will result in an average of the structures encountered, with some loss of definition. Crystals cooled to near liquid-N2 temperature typically show a greatly reduced rate of radiation damage, often to the extent that it is no longer an issue of concern. The protection from radiation damage was noted early on. Petsko (1975)[link] observed numerous cases of this effect. A noteworthy example is the successful prevention of radiation damage to crystals of ribosome particles (Hope et al., 1989[link]).

Radiation damage appears to be related to the formation of free radicals. At sufficiently low temperature, two effects can influence the rate of damage: movement of the radicals is hampered, and the activation energy for reaction is not available. A revealing observation has been described by Hope (1990)[link]. A crystal that had been exposed to synchrotron radiation for many hours at 85 K showed no overt signs of radiation damage. However, while the crystal was being warmed toward room temperature, it suddenly turned black and curled up like a drying leaf. More commonly, crystals turn yellow under X-ray irradiation, and bubbles and cracks appear on warming. The rate of free-radical formation would be affected little by temperature, so that when sufficient mobility and activation energy become available, the stored radicals will react. Mechanical stability of the crystal mount

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The mechanical stability of samples is also of concern. Crystals mounted in capillaries and kept wet have a tendency to move, giving rise to difficulties with intensity measurements. A crystal at cryotemperature is rigidly attached to its mount; slippage is impossible. Effect on resolution

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The effects on radiation damage and mechanical stability are clear-cut, and provide the main reasons for using cryotechniques. Resolution can also be affected, but the connection between temperature and resolution is neither simple nor obvious. If low resolution is the result of rapid radiation damage, lowering the temperature can lead to much improved resolution. However, if low resolution is mainly caused by inexact replication from one unit cell to another, lowering the temperature may have little effect on resolution. If the mosaic spread in the crystal increases upon cooling, resolution may even deteriorate.

In a model proposed by Hope (1988[link]), a relationship between resolution r and temperature T is given by [r_{2} = r_{1}[(B_{0} + bT_{2})/(B_{0} + bT_{1})]^{1/2}.] Here [r_{1}] is the resolution at [T_{1}], [r_{2}] is the resolution at [T_{2}], [B_{0}] is the value of B at [T = 0] and b is a proportionality constant. The underlying assumption is that for any given temperature, the temperature factor [i.e. [\exp(-B\sin^{2}\theta/\lambda^{2})]] at the resolution limit has the same value; thus the effects of scattering factors and Lp factors are ignored. We see that if [B_{0}] is the predominant term, lowering T will not have much effect, whereas for small [B_{0}] (a relatively well ordered structure) the effect of T on r can be large. For example, if the room-temperature resolution is 1.5 Å, the resolution at 100 K can be around 1 Å, but if the room-temperature resolution is around 3 or 4 Å, little change can be expected. A qualitative assessment of these effects was clearly stated by Petsko (1975)[link].


Hope, H.(1988). Cryocrystallography of biological macromolecules: a generally applicable method. Acta Cryst. B44, 22–26.Google Scholar
Hope, H. (1990). Cryocrystallography of biological macromolecules at ultra-low temperature. Annu. Rev. Biophys. Biophys. Chem. 19, 107–126.Google Scholar
Hope, H., Frolow, F., von Böhlen, K., Makowski, I., Kratky, C., Halfon, Y., Danz, H., Webster, P., Bartels, K. S., Wittmann, H. G. & Yonath, A. (1989). Cryocrystallography of ribosomal particles. Acta Cryst. B45, 190–199.Google Scholar
Petsko, G. A. (1975). Protein crystallography at sub-zero temperatures: cryoprotective mother liquors for protein crystals. J. Mol. Biol. 96, 381–392.Google Scholar

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