Radiation damage in sub-Ångström resolution macromolecular crystallography: a low-dose study

Sub-Ångström macromolecular crystallography allows the construction of structural models without any prior chemical knowledge and stereochemical restraints, making it possible to visualize deviations from peptide planarity, distortions of the planarity of aromatic ring systems and subtle alternate conformation networks. It also holds the promise of observing aspherical density features and other quantum-mechanical phenomena. However, the degree to which radiation damage might affect the details that can be discerned from biological structures at such resolutions has remained unknown. To address this, we report here the first study of radiation-damage effects at sub-Ångström resolution on Pyrococcus abyssi rubredoxin. Our data, collected at 100 K, indicate that the oxidized (Fe³⁺) state of the Fe atom is preserved to a large degree at a dose of 50 kGy, whereas at 1 MGy it is partially reduced to the Fe²⁺ state. Isomorphous difference maps reveal extensive conformational changes at 1 MGy that are most likely coupled to the reduction of Fe³⁺. At 1 MGy, hydrogen densities that are visible at 50 kGy are preserved, but are distinctly blurred by dose. These findings suggest that the `global' radiation damage in reciprocal space and the associated blurring of electron density in real space proceed through small structural changes that are much more extended than the local damage at specific sites which is normally seen at lower resolution. Thus, the `global' and `specific' damage could be viewed as two sides of the same coin. Our study highlights the unique benefits of using low-dose data-collection protocols on large crystals fully bathed in a large `top-hat' beam with a uniform fluence profile for accurate sub-Ångström structural investigations on macromolecules, as only this experimental configuration can deliver the uniform spatial distribution of dose necessary to resolve the fine details of progressive radiation damage: a capability for which we propose the term `resolution in dose'.