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

International Tables for Crystallography (2006). Vol. F, ch. 9.1, pp. 191-192   | 1 | 2 |

Section 9.1.12. Radiation damage

Z. Dautera* and K. S. Wilsonb

aNational Cancer Institute, Brookhaven National Laboratory, NSLS, Building 725A-X9, Upton, NY 11973, USA, and bStructural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, England
Correspondence e-mail:

9.1.12. Radiation damage

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All crystals irradiated with X-rays absorb at least a fraction of the radiation, resulting in damage to the sample (Henderson, 1990[link]). The energy from the absorbed photons may initially result in the disruption of chemical bonds, before being eventually dissipated as thermal energy. For well ordered small-molecule crystals the lattice is close packed and the effects arising from the absorbed photons are restricted to the immediate environment of the absorption event, so-called primary damage. Only when a substantial fraction of the crystal has been affected do cooperative effects set in.

In contrast, roughly 50% of a macromolecular crystal is disordered aqueous solvent (Matthews, 1968[link]). At room temperature this allows a secondary mechanism of radiation damage, resulting from diffusion of radicals and ions produced at the primary absorption site that affects chemical moieties at positions remote from this site. The details of this process remain poorly understood but are related to the extremely damaging effects of X-rays on biological tissue. A consequence of this damage is that degradation of the crystal order continues even after the irradiation is stopped or interrupted. For collection of data at room temperature from protein crystals mounted in capillaries, secondary damage contributes significantly to the rate of deterioration of the diffraction pattern. One of the gains of the early applications of SR was that it allowed recording of data to proceed ahead of the effects of secondary damage, increasing the effective, if not the absolute, lifetime of the crystal in the X-ray beam. An experiment often required several crystals, all of which showed the effects of temporal decay in their recorded intensities, which needed to be merged to provide complete data. Cryogenic freezing

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In the early 1990s, the introduction of protein-data collection at cryogenic temperatures, using so-called flash freezing, was a major breakthrough (Garman & Schneider, 1997[link]; Rodgers, 1997[link]). Flash-frozen crystals largely prevented the effects of secondary damage. On the X-ray sources then available, it was in most cases possible to record complete data from a single sample without significant degradation of the diffraction, enormously simplifying the strategy of data collection and merging.

The techniques of macromolecular cryocrystallography have advanced so rapidly that almost all data are currently collected from frozen samples. The key aspects of flash freezing are addressed in Part 10[link] . The prolonged life of the sample and modest rates of data acquisition, even at second-generation SR sources with imaging plates, allowed enough time for careful analysis of the initial images and optimization of the strategy.

A second major advantage of cryogenic freezing is that it allows crystals to be reused after initial data have been recorded. Two examples show the usefulness of this approach. Firstly, when screening the binding of heavy atoms for phase determination or ligands for complex formation, data can first be recorded to the minimum resolution needed to determine whether the binding is successful. Secondly, a series of frozen crystals can be screened for their degree of order in the home laboratory, and the best stored and retained for subsequent improved collection either in the home laboratory or at a synchrotron site. The ability to transport frozen crystals has proved invaluable in this respect, and leads to optimal use of synchrotron resources. Ultra high intensity SR sources

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The advent of third-generation SR sources and insertion devices has led to X-ray beams of unprecedented intensity, for example at the ESRF or APS. At the time of writing, the first of these beamlines have only recently been commissioned and it is hard to give a precise evaluation of their implications for data-collection strategy. Hence the experience to date is somewhat anecdotal and is not based on published reports.

The speed of data collection can be of the order of 1 second per 1° rotation. In association with CCD detectors able to read out images within a few seconds, this means that a complete data set can be obtained in a few minutes. At first sight this would seem to have solved the problem of macromolecular data collection, as such speeds should allow recording of highly redundant accurate data to the highest resolution in a tractable time. However, with these ultra high intensities it appears that a new element of damage can occur. The useful active exposure lifetime of typical crystals seems to be around five minutes, with substantial degradation of the diffraction pattern ensuing even for cryogenically frozen crystals. This may be a limitation of the rate at which heat resulting from the absorption of photons can be dissipated, with local heat gradients perhaps being the factor responsible for the disruption of the crystal order.

This effect suggests that adopting strategies for choosing the optimal starting point of rotation in the minimal total rotation approach for complete data may once more be vital. Using current software this can be achieved in a matter of minutes. It is worth sacrificing this time for the sake of data quality.


Garman, E. F. & Schneider, T. R. (1997). Macromolecular cryocrystallography. J. Appl. Cryst. 30, 211–237.
Henderson, R. (1990). Cryo protection of protein crystals against radiation damage in electron and X-ray diffraction. Proc. R. Soc. London Ser. B, 241, 6–8.
Matthews, B. W. (1968). Solvent content in protein crystals. J. Mol. Biol. 33, 491–497.
Rodgers, D. W. (1997). Practical cryocrystallography. Methods Enzymol. 276, 183–203.

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