Two-dimensional crystals of the Escherichia coli DNA gyrase B subunit were obtained upon specific interactions with novobiocin linked phospholipid films. A three-dimensional surface model of the protein was generated by analysing images of tilted negatively stained crystals. The structure showed, at 2.5 to 3.0 nm resolution, two elongated arms organised as a V-shaped protein: the bottom of the V contains the novobiocin binding site, and the extremities of the arms mediate protein-protein interactions between the two monomers in the unit cell. Image analysis of frozen hydrated two-dimensional crystals resulted in a 1.0 nm resolution projection map that shows structural elements not revealed with negative staining. Electron microscopic structural data were compared with the crystallographic structure of the 43 kDa N-terminal fragment of the B subunit complexed with a non hydrolysable ATP analogue.