The 'fingerprint' that X-rays can leave on structures

Background: Exposure of biomacromolecules to ionising radiation results in damage that is initiated by free radicals and progresses through a variety of mechanisms. A widely used technique to study the three-dimensional struc tures of biomacromolecules is crystallography, which makes use of ionising X-rays. It is crucial to know to what extent structures determined using th is technique might be biased by the inherent radiation damage. Results: The consequences of radiation damage have been investigated for th ree dissimilar proteins. Similar results were obtained for each protein, at omic B factors increase, unit-cell volumes increase, protein molecules unde rgo slight rotations and translations, disulphide bonds break acid decarbox ylation of acidic residues occurs. All of these effects introduce non-isomo rphism. The absorbed dose in these experiments can be reached during routin e data collection at undulator beamlines of third generation synchrotron so urces. Conclusions: X-rays can leave a 'fingerprint' on structures, even at cryoge nic temperatures. Serious non-isomorphism can be introduced, thus hampering multiple isomorphous replacement (MIR) and multiwavelength anomalous dispe rsion (MAD) phasing methods. Specific structural changes can occur before t he traditional measures of radiation damage have signalled it. Care must be taken when assigning structural significance to features that might easily be radiation-damage-induced changes. It is proposed that the electron-affi nic disulphide bond traps electrons that migrate over the backbone of the p rotein, and that the sidechains of glutamic acid and aspartic acid donate e lectrons to nearby electron holes and become decarboxylated successively. T he different disulphide bonds in each protein show a clear order of suscept ibility, which might well relate to their intrinsic stability.