Streptavidin forms two-dimensional crystals when specifically bound to layers of biotinylated lipids at the air/water interface. The three-dimensional structure of streptavidin determined from the crystals by electron crystallography corresponds well with the structure determined by x-ray crystallography. Comparison of the electron and x-ray crystallographic structures reveals the occurrence of free biotin-binding sites on the surface of the two-dimensional crystals facing the aqueous solution. The free biotin-binding sites could be specifically labeled with biotinylated ferritin. The streptavidin/biotinylated lipid system may provide a general approach for the formation of two-dimensional crystals of biotinylated macromolecules.

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The B subunit of cholera toxin forms twodimensional crystals when bound to its membrane receptor, ganglioside GM1, in phospholipid layers. A rectangular crystal lattice gives diffraction extending to 15-A resolution in negative stain, and image-processing of electron micrographs reveals a ring of five protein densities. The diameter of the central hole and the outer diameter of the ring are about 20 and 60 A, respectively. These data are consistent with a pentameric, doughnut-shaped structure of the B subunit that lies flat on a membrane surface. A hexagonal crystal lattice is obtained as well, and results of image processing and chemical crosslinking allow two interpretations: ('l the B subunit may exist in both pentameric and hexameric forms or, more likely, (it) the hexagonal lattice may represent a disordered or liquid crystalline form, in which a pentamer undergoes rotational averaging about its 5-fold axis.The clinical symptoms of cholera are attributable to the effects of a toxin produced by Vibrio cholerae (1-3). Cholera toxin is composed of two subunits, A, cleaved in the active form into A1 (Mr 22,000) and A2 (Mr 5,000) fragments, and B (Mr 11,600) (4-7). The B subunit forms an oligomer, which is generally believed, on the basis of ultracentrifugation and chemical crosslinking studies, to be a pentamer (5,8,9), although other evidence is suggestive of tetrameric or hexameric forms (9, 10). The action of cholera toxin on susceptible mammalian cells begins with binding of the B subunit to a cell surface receptor, the monosialoganglioside GM1, with an association constant of about 109 M-1 (11). The oligosaccharide moiety of GM1 bears the determinants for this interaction (12). Binding is followed by a lag period, during which the Al fragment penetrates the plasma membrane. Finally, Al catalyzes the ADP-ribosylation of adenylate cyclase (13)(14)(15)(16)(17)(18)(19). The events during the lag period and the mechanism of penetration of Al present major challenges for the understanding of toxin action.Although the chemical characterization of cholera toxin has been extensive (4,5,(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29), there is a lack of the three-dimensional structural information needed to explain the membrane-penetration process. "V-shaped" and "ringlike" particles, 70-105 A in diameter, have been seen in electron micrographs of cholera toxin and B-subunit oligomers (20). Single crystals of cholera toxin suitable for x-ray diffraction analysis have been obtained (30), but the structure has not been reported. We describe here the use of the lipid-layer crystallization technique (31) for imaging B oligomers. The technique exploits binding to a lipid ligand in mono-or multilayers to orient and concentrate a macromolecule. Diffusion within the lipid layers allows the formation of two-dimensional crystals amenable to structure determination by electron microscopy and image processing. This approach gives lower resolution than x-ray diffraction, but it has the advantages that crystals form ...

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The B1 subunit of ribonucleotide reductase formed two-dimensional crystals when bound to and effector nucleotide linked to lipids in planar layers at the air/water interface. The effector lipid consisted of dATP coupled through the gamma-phosphoryl group and an epsilon-aminocaproyl linker to phosphatidylethanolamine. Two-dimensional crystals of B1 reductase, like those of antibodies and cholera toxin obtained previously, formed under physiologic conditions of pH and ionic strength, with no precipitant added to the solution. There was, however, a requirement for dTTP in the solution, presumably to ensure binding of the dATP-lipid at only one of two effector sites on the enzyme. Diffraction from the crystals extended to 18-A resolution in negative stain, with unit cell parameters a = 110 A, b = 277 A, and gamma = 90 degrees. Image analysis revealed the B1 dimer as a pair of roughly cylindrical objects, each 105-109 A in length and 31-34 A in diameter.