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

International Tables for Crystallography (2012). Vol. F, ch. 9.1, pp. 225-226   | 1 | 2 |

Section 9.1.12. Radiation damage

Z. Dautera* and K. S. Wilsonb

aNCI Frederick & Argonne National Laboratory, Building 202, Argonne, IL 60439, USA, and bYork Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5YW, 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 which affect 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 vitrification

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In the early 1990s, the introduction of protein-data collection at cryogenic temperatures, using so-called flash cooling, was a major breakthrough (Garman & Schneider, 1997[link]; Rodgers, 1997[link]; Garman & Owen, 2006[link]). Such vitrification of 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.

Almost all data are currently collected from vitrified samples (see Part 10[link] ). The prolonged life of the sample and modest rates of data acquisition, even at second-generation SR sources with imaging plates, allow enough time for careful analysis of the initial images and optimization of the strategy.

A second major advantage of cryogenic data collection 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 vitrified 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 vitrified crystals has proved invaluable in this respect, and leads to optimal use of synchrotron resources. High-intensity third-generation 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. 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 such high intensities it appears that the effects of radiation damage are significant and result in specific effects on susceptible parts of the structure. The useful active exposure lifetime of typical crystals seems to be around five minutes, with substantial degradation of the diffraction pattern ensuing even for vitrified crystals. The first manifestation of radiation damage is the disruption of disulfide bridges and decarboxylation of aspartates and glutamates. This effect means appropriate strategies for selecting the optimal starting point of rotation in order to minimize the total rotation required for collection of complete data are once more essential. Several strategy programs, such as BEST (Popov & Bourenkov, 2003[link]; Bourenkov & Popov, 2006[link]), now permit this to be done effectively. Correcting data for the effects of radiation damage

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The overall effect of radiation damage is that the higher-resolution intensities decrease faster than those at low resolution. This effect is largely taken into account by the relative B factors applied to individual images during data scaling and merging by the major data-reduction programs.

However, such scaling does not allow for the effect of specific structural damage (e.g. the S–S bridges and carboxylic groups) on individual reflection intensities. A method to deal with this has been proposed by Diederichs et al. (2003)[link]. This is based on a zero-dose extrapolation of intensities and requires that a timestamp be attached to each individual intensity measurement. Such a timestamp has also been used to assist in estimation of phases by SHARP (Schiltz et al., 2004[link]).


Bourenkov, G. P. & Popov, A. N. (2006). A quantitative approach to data-collection strategies. Acta Cryst. D62, 58–64.
Diederichs, K., McSweeney, S. & Ravelli, R. B. G. (2003). Zero-dose extrapolation as part of macromolecular synchrotron data reduction. Acta Cryst. D59, 903–909.
Garman, E. F. & Owen, R. L. (2006). Cryocooling and radiation damage in macromolecular crystallography. Acta Cryst. D62, 32–47.
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.
Popov, A. N. & Bourenkov, G. P. (2003). Choice of data-collection parameters based on statistic modelling. Acta Cryst. D59, 1145–1153.
Rodgers, D. W. (1997). Practical cryocrystallography. Methods Enzymol. 276, 183–203.
Schiltz, M., Dumas, P., Ennifar, E., Flensburg, C., Paciorek, W., Vonrhein, C. & Bricogne, G. (2004). Phasing in the presence of severe site-specific radiation damage through dose-dependent modelling of heavy atoms. Acta Cryst. D60, 1024–1031.

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