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.

Influenza virus neuraminidase catalyses the cleavage of terminal sialic acid, the viral receptor, from carbohydrate chains on glycoproteins and glycolipids. We present the crystal structure of the enzymatically active head of influenza B virus neuraminidase from the strain B/Beijing/1/87. The native structure has been refined to a crystallographic R‐factor of 14.8% at 2.2 A resolution and its complex with sialic acid refined at 2.8 A resolution. The overall fold of the molecule is very similar to the already known structure of neuraminidase from influenza A virus, with which there is amino acid sequence homology of approximately 30%. Two calcium binding sites have been identified. One of them, previously undescribed, is located between the active site and a large surface antigenic loop. The calcium ion is octahedrally co‐ordinated by five oxygen atoms from the protein and one water molecule. Sequence comparisons suggest that this calcium site should occur in all influenza A and B virus neuraminidases. Soaking of sialic acid into the crystals has enabled the mode of binding of the reaction product in the putative active site pocket to be revealed. All the large side groups of the sialic acid are equatorial and are specifically recognized by nine fully conserved active site residues. These in turn are stabilized by a second shell of 10 highly conserved residues principally by an extensive network of hydrogen bonds.

The three-dimensional structure of the rat neonatal Fc receptor (FcRn) is similar to the structure of molecules of the major histocompatibility complex (MHC). The counterpart of the MHC peptide-binding site is closed in FcRn, making the FcRn groove incapable of binding peptides. A dimer of FcRn heterodimers seen in the crystals may represent a receptor dimer that forms when the Fc portion of a single immunoglobulin binds. An alternative use of the MHC fold for immune recognition is indicated by the FcRn and FcRn/Fc co-crystal structures.

Myrosinase, an S-glycosidase, hydrolyzes plant anionic 1-thio-␤-D-glucosides (glucosinolates) considered part of the plant defense system. Although O-glycosidases are ubiquitous, myrosinase is the only known Sglycosidase. Its active site is very similar to that of retaining O-glycosidases, but one of the catalytic residues in O-glycosidases, a carboxylate residue functioning as the general base, is replaced by a glutamine residue. Myrosinase is strongly activated by ascorbic acid. Several binary and ternary complexes of myrosinase with different transition state analogues and ascorbic acid have been analyzed at high resolution by x-ray crystallography along with a 2-deoxy-2-fluoro-glucosyl enzyme intermediate. One of the inhibitors, D-gluconhydroximo-1,5-lactam, binds simultaneously with a sulfate ion to form a mimic of the enzyme-substrate complex. Ascorbate binds to a site distinct from the glucose binding site but overlapping with the aglycon binding site, suggesting that activation occurs at the second step of catalysis, i.e. hydrolysis of the glycosyl enzyme. A water molecule is placed perfectly for activation by ascorbate and for nucleophilic attack on the covalently trapped 2-fluoroglucosyl-moiety. Activation of the hydrolysis of the glucosyl enzyme intermediate is further evidenced by the observation that ascorbate enhances the rate of reactivation of the 2-fluoro-glycosyl enzyme, leading to the conclusion that ascorbic acid substitutes for the catalytic base in myrosinase.Glucosinolates are anionic ␤-D-S-glucosides found prominently in plants of the genus Brassica (cabbage, mustard, rapeseed, and other Cruciferae). They constitute a large family of S-glucosides that differ by their aglycon (Ref. 1 and Fig. 1a). The same plants produce myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1), an S-glucosidase hydrolyzing glucosinolates. Myrosinase and glucosinolates are stored in different compartments of the plant, especially in the seeds. Mixing of enzyme and substrate (for example through mastication) induces glucosinolate hydrolysis. The biological function of myrosinase and glucosinolates is only partly elucidated; it has been suggested that they represent a defense system of the plant. Glucosinolates may as well serve to store inactive precursors of hormones such as 3-indolylacetic acid. 3-Indolyl acetonitrile and related indoles (2) are released from indolyl glucosinolates by myrosinase (3). Cleavage of indol-3-ylmethyl glucosinolate (glucobrassicin) by myrosinase in the presence of ascorbic acid produces ascorbigen, a condensation product of ascorbic acid with 3-hydroxymethylindole (4). Thus, the myrosinase system could be involved in the storage and in the inactivation of ascorbic acid. A detailed review on myrosinase is given by Bones and Rossiter (3).Myrosinase hydrolyzes the S-glycosides with retention of the anomeric configuration (5). Retaining glycosidases operate by a double displacement at the anomeric center promoted by two carboxylic residues acting as acid/base and as nucleophile, respec...

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The amino-terminal domain of influenza A virus matrix protein (residues 1-164) was crystallized at pH 7 into a new crystal form in space group P1. This packing of the protein implies that M1(1-164) was monomeric in solution when it crystallized. Otherwise, the structure of the M1 fragment in the pH 7 crystals was the same as the monomers in crystals formed at pH 4 where crystal packing resulted in dimer formation [B. Sha and M. Luo, 1997, Nature Struct. Biol. 4, 239-244]. Analysis of intact M1 protein, the N-terminal domain, and the remaining C-terminal fragment (residues 165-252) in solution also showed that the N-terminal domain was monomeric with the same dimensions as determined from the crystal structure. Intact M1 protein was also monomeric but with an elongated shape due to the presence of the C-terminal part. Circular dichroism showed that the C-terminal part of M1 contained helical structure. A model for soluble M1 is presented, based on the assumption that the C-terminal domain is spherical, in which the N- and C-terminal domains are connected by a linker sequence which is available for proteolytic attack.