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. 10.1, pp. 242-243   | 1 | 2 |

Section Suppression of radiation damage

H. Hopea* and S. Parkinb

aDepartment of Chemistry, University of California, Davis, One Shields Ave, Davis, CA 95616–5295, USA, and bDepartment of Chemistry, University of Kentucky, Lexington, Kentucky, USA
Correspondence e-mail: Suppression of radiation damage

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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 invariably 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 concomitant 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 major concern. The protection from radiation damage was noted early on (Haas & Rossman, 1970[link]) and numerous cases were observed by Petsko (1975)[link]. 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 most commonly initiated by photoelectrons and propagated by their inelastic scattering from nearby atoms, creating positively charged species and a cascade of secondary electrons (Garman & Nave, 2009[link]). These can further interact with either protein or with water to form reactive, charged species and free radicals. At sufficiently low temperature, two effects can influence the rate of damage: movement of the reactive species is impeded and the activation energy for reaction is not available. A revealing observation has been described by Hope (1990)[link], where a crystal that had been exposed to syn­chrotron radiation for many hours at 85 K showed no overt signs of radiation damage, but as 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 little affected by temperature, so that when sufficient mobility and activation energy become available, the stored radicals will react.

There is a general consensus that radiation damage, even in high-flux synchrotron beams, can be slowed by cooling to liquid-helium temperatures. The extent of radiation damage is expected to depend on the nature of the macromolecule, and literature examples clearly illustrate this. Meents et al. (2007)[link] have reported an extensive study of insulin and holoferritin at 15 and 90 K. They observed statistically significant but relatively small reductions in radiation damage; for holoferritin, 23% less damage at the lower temperature, and for insulin about 6%. In a study of Streptomyces rubiginosus D-xylose isomerase, Chinte et al. (2007)[link] found a lifetime extension of 25% at 8 K compared to data collection at 100 K. Corbett et al. (2007)[link] compared results from data collected at ~40 and at 110 K in a study of the metalloprotein putida­redoxin. They report that radiation-induced photoreduction at 110 K resulted in misleading structure interpretation. At 40 K the photoreduction did not occur. The damage mechanism in this case is related to a change in oxidation state of a central metal atom. The authors made a strong argument for measuring data for metalloproteins at the lowest possible temperature. Chinte et al. (2007)[link] pointed out that the additional cost of liquid-helium-temperature measurements compared to 90–100 K measurements is small, and that the advantages can significantly outweigh the increased cost.

In recent years, the ability to calibrate X-ray beam intensity, to calculate reasonable approximations to accumulated radiation dose and to assess crystal degradation by means of various spectroscopies (e.g. UV–visible, IR, Raman, XAFS, XANES) in concert with diffraction has brought dramatic advances in understanding radiation damage by X-rays. A concise account has been given by Garman & Nave (2009)[link]. Useful suggestions for avoidance of radiation damage, including cases where multiple crystals are required, have been given by Holton (2009)[link]. Briefly, knowledge of beamline photon flux density (photons µm−2 s−1), the dose ratio (a function of crystal composition and X-ray wavelength), the crystal shape and the cross section intensity profile of a given X-ray beam allows an estimate of the maximum tolerable exposure time for a particular crystal.


Chinte, U., Shah, B., Chen, Y.-S., Pinkerton, A. A., Schall, C. A. & Hanson, B. L. (2007). Cryogenic (<20 K) helium cooling mitigates radiation damage to protein crystals. Acta Cryst. D63, 486–492.
Corbett, M. C., Latimer, M. J., Poulos, T. L., Sevrioukova, I. F., Hodgson, K. O. & Hedman, B. (2007). Photoreduction of the active site of the metalloprotein putidaredoxin by synchrotron radiation. Acta Cryst. D63, 951–960.
Garman, E. F. & Nave, C. (2009). Synchrotron radiation damage in protein crystals examined under various conditions by different methods. J. Synchrotron Rad. 16, 129–132.
Haas, D. J. & Rossmann, M. G. (1970). Crystallographic studies on lactate dehydrogenase at −75 °C. Acta Cryst. B26, 998–1004.
Holton, J. M. (2009). A beginner's guide to radiation damage. J. Synchrotron Rad. 16, 133–142.
Hope, H. (1990). Cryocrystallography of biological macromolecules at ultra-low temperature. Annu. Rev. Biophys. Biophys. Chem. 19, 107–126.
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.
Meents, A., Wagner, A., Schneider, R., Pradervand, C., Pohl, E. & Schulze-Briese, C. (2007). Reduction of X-ray-induced radiation damage of macromolecular crystals by data collection at 15 K: a systematic study. Acta Cryst. D63, 302–309.
Petsko, G. A. (1975). Protein crystallography at sub-zero temperatures: cryoprotective mother liquors for protein crystals. J. Mol. Biol. 96, 381–392.

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