Introduction. In response to fungal invasion and other signals, plants accumulate a number of proteins that are involved in defense against pathogens. 1 Among these proteins, pathogenesis-related (PR) proteins are grouped into families based on primary structure, serological relatedness, and enzymatic and biological activities. 2 Osmotin is a 24 kDa protein belonging to the PR-5 protein family whose members are homologous to the sweet-tasting protein thaumatin. Osmotin and other PR-5 proteins were shown to have antifungal activity in vitro against a broad range of fungi, including several plant pathogens. 1 PR-5 proteins can be categorized into three subclasses based on their isoelectric point (pI): acidic, neutral and basic. 3 Comparative analysis of the primary structure of osmotin, which is a basic PR-5 protein from tobacco, and several other PR-5 proteins reveal several interesting features. 4 The alanine located at the cleavage site of N-terminal leader sequence and 16 cysteine residues, which are distributed throughout the protein and linked via formed disulfide-bridges, are conserved spatially among virtually all PR-proteins. 4 The fungal growth inhibition by osmotin and zeamtin, a maize PR-5 protein, is correlated with plasma membrane permeabilization and dissipation of the plasma membrane potential, 5,6 suggesting a physical interaction between PR-5 proteins and the plasma membrane of sensitive fungi. In contrast, it is proposed that specific target receptors on the membrane determine the sensitivity or resistance to PR-5 proteins. 7 However, the precise mechanism by which osmotin interacts with specific fungal pathogens and mediates plasma membrane permeability has not been clearly elucidated. Recent genetic and biochemical data indicate that osmotin utilizes a signal transduction pathway in yeast to increase the susceptibility of a target fungus to its cytotoxic effects. 8 To understand the structural basis of antifungal activity of osmotin, we have determined the crystal structure of tobacco osmotin at 2.3 Å resolution and compared its structure with other antifungal proteins and thaumatin.Materials and Methods. Osmotin was purified from salt-adapted tobacco cell suspension cultures (Nicotiana tabacum L. var. Wisconsin 38) to apparent homogeneity as described. 9 Lyophilized osmotin was dissolved in 25 mM N-2-hydroxyethylpiperazine-NЈ-2-ethanesulfonic acid (HEPES; pH 7.5) to the concentration of 7 mg/mL. Crystals were obtained by the vapor diffusion method at 22°C in hanging drops containing a 1:1 mixture of a protein solution with reservoir solution (0.1 M Tris-HCl, pH 8.5, 0.4 M lithium sulfate, 0.06 M nickel chloride). Osmotin crystals belong to space group P1 with unit cell dimensions of a ϭ 41.78 Å, b ϭ 41.79 Å, c ϭ 59.56 Å, ␣ ϭ 100.84°,  ϭ 92.41°, ␥ ϭ 96.57°. There are two monomers in an asymmetric unit, with the corresponding V m of 2.27 Å 3 /Da and the solvent content of 45%. The crystal diffracted to 2.3 Å resolution using synchrotron X-rays at Brookhaven National Laboratory. A total of 26,5...