Radiation damage of crystalline biological samples has become a daily problem on the undulator beamlines of third-generation sources, even at cryogenic temperatures. Whereas many crystalline samples show a clear increase in resolution when using the ESRF undulator beamlines, this increase cannot always be maintained throughout the data collection because of the problem of radiation damage. Until recently, it was generally believed that radiation damage is non-specific, in that it would only affect the resolution at which the structure could be studied but would not affect the structure itself. However, three studies [1,2,3] performed at the ESRF beamlines ID14-3 and ID14-4 have shown that highly specific changes can occur. Disulphide bonds fragment when the absorbed dose increases, and acidic residues become decarboxylated. Thus, X-rays can leave an indelible "fingerprint" on the structure (Figure 7).

Figure 7
Fig. 7: A difference electron density map showing only one sulphur atom of a disulphide bridge. This bridge was intact in the native protein, but has been ruptured as a result of radiation damage. 

The onset of this damage can occur even before clear changes in the diffractive power of the crystal are observed, and it is likely that many published structures have not taken this into account. New and better-suited criteria have now been introduced that are indicative of the X-ray "fingerprint" and these include changes in the unit cell volume that occur even during the early stages of radiation damage.

Secondary damage, mediated by mobile electron centres and holes, both coming from the protein ("direct") and the solvent area ("indirect"), seems to play a crucial role. The one electron reduction of a disulphide bond to RSSR·- is well known from radiolysis studies and occurs with a very high rate constant (1.5x1010 L mol­1 s­1 at room temperature and pH 6.2). This reaction also occurs during experiments with crystals irradiated at 100 K, and the disulphide radical may further react to form others species such as RS·, RSH and RS-. The decarboxylation of acidic residues may proceed via the transfer of an electron hole to the carboxyl group, which in turn leads to the formation of CO2 and a carbon centred radical.

Radiation damage induced by X-rays is not just an experimental problem. The clear order that is observed in the susceptibility to reduction of different disulphide bonds within a sample could be a simple measure of the environment of the disulphide bond. There is a range of more than 11 orders of magnitude in stability among disulphide bonds within different proteins as measured by Kox, which gives the stability of a disulphide bond to reduction by glutathione. There may be a clear correlation between the susceptibility to radiation damage and Kox. It has been observed that during bond breakage one of the sulphur atoms remains relatively fixed in position, whereas the other is labile and this could be of direct relevance to understanding the dynamics of enzymes that require labile disulphide bonds for activity, such as thioredoxin and DsbA. Recently it has also been shown that specific changes in the macromolecular structures induced by radiation damage may be of real use for phasing macromolecular structures.

[1] M. Weik, R.B.G. Ravelli, G. Kryger, S. McSweeney, M.L. Raves, M. Harel, P. Gros, I. Silman, J. Kroon and J.L. Sussman, PNAS, 97(2), 623-628 (2000).
[2] R.B.G. Ravelli and S. McSweeney, Structure, 8, 315-328 (2000).
[3] W.P. Burmeister, Acta Cryst., D56, 328-341 (2000).

R.B.G. Ravelli (a) and S. McSweeney (b).
(a) EMBL Grenoble Outstation (France)
(b) ESRF