The high intensity of third‐generation X‐ray sources, along with the development of cryo‐cooling of protein crystals at temperatures around 100 K, have made it possible to extend the diffraction limit of crystals and to reduce their size. However, even with cryo‐cooled crystals, radiation damage becomes a limiting factor. So far, the radiation damage has manifested itself in the form of a loss of overall diffracted intensity and an increase in the temperature factor. The structure of a protein (myrosinase) after exposure to different doses of X‐rays in the region of 20 × 1015 photons mm−2 has been studied. The changes in the structure owing to radiation damage were analysed using Fourier difference maps and occupancy refinement for the first time. Damage was obvious in the form of breakage of disulfide bonds, decarboxylation of aspartate and glutamate residues, a loss of hydroxyl groups from tyrosine and of the methylthio group of methionine. The susceptibility to radiation damage of individual groups of the same kind varies within the protein. The quality of the model resulting from structure determination might be compromised owing to the presence of radiolysis in the crystal after an excessive radiation dose. Radiation‐induced structural changes may interfere with the interpretation of ligand‐binding studies or MAD data. The experiments reported here suggest that there is an intrinsic limit to the amount of data which can be extracted from a sample of a given size.

The structure of myrosinase shows features which illustrate the adaptation of the plant enzyme to the dehydrated environment of the seed. The catalytic mechanism of myrosinase is explained by the excellent leaving group properties of the substrate aglycons, which do not require the assistance of an enzymatic acid catalyst. The replacement of the general acid/base glutamate of O-glycosidases by a glutamine residue in myrosinase suggests that for hydrolysis of the glycosyl-enzyme, the role of this residue is to ensure a precise positioning of a water molecule rather than to provide general base assistance.

contributed equally to this work During in¯uenza virus infection, viral ribonucleo proteins (vRNPs) are replicated in the nucleus and must be exported to the cytoplasm before assembling into mature viral particles. Nuclear export is mediated by the cellular protein Crm1 and putatively by the viral protein NEP/NS2. Proteolytic cleavage of NEP de®nes an N-terminal domain which mediates RanGTP-dependent binding to Crm1 and a Cterminal domain which binds to the viral matrix protein M1. The 2.6 A Ê crystal structure of the C-terminal domain reveals an amphipathic helical hairpin which dimerizes as a four-helix bundle. The NEP±M1 interaction involves two critical epitopes: an exposed tryptophan (Trp78) surrounded by a cluster of glutamate residues on NEP, and the basic nuclear localization signal (NLS) of M1. Implications for vRNP export are discussed.