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Micrographs of mouse liver gap junctions, isolated with detergents, and negatively stained with uranyl acetate, have been recorded by low-irradiation methods. Our Fourieraveraged micrographs of the hexagonal junction lattice show skewed, hexameric connexons with less stain at the threefold axis than at the six indentations between the lobes of the connexon image. These substructural features, not clearly observed previously, are acutely sensitive to irradiation. After an electron dose less than that normally used in microscopy, the image is converted to the familiar doughnut shape, with a darkly stained center and a smooth hexagonal outline, oriented with mirror symmetry in the lattice. Differences in appearance among 25 reconstructed images from our low-irradiation micrographs illustrate variation in staining of the connexon channel and the space between connexons. Consistently observed stain concentration at six symmetrically related sites ~34 tk from the connexon center, 8 ° to the right or left of the (1, 1) lattice vector may reveal an intrinsic asymmetric feature of the junction structure. The unexpected skewing of the six-lobed connexon image suggests that the pair of hexagonal membrane arrays that form the junction may not be structurally identical. Because the projected image of the connexon pair itself appears mirror symmetric, each pair may consist of two identical connexon hexamers related by local (noncrystallographic) twofold axes in the junctional plane at the middle of the gap. All connexons may be chemically identical, but their packing in the hexagonal arrays on the two sides of the junction appears to be nonequivalent.Gap junctions in liver (13, 28) and many other tissues (5, 10) are built of connexon units (6) hexagonally arrayed with crystaUike regularity in the pair of connected cell membranes. Makowski et al. (21) proposed a model for the junction structure based on data from x-ray diffraction, electron microscopy and chemical analysis in which the connexon units were pictured as symmetric hexamers of identical connexin molecules that are equivalently paired across the gap with dihedral symmetry in the two-sided hexagonal plane group (p622; see reference 16). This model predicts that, as normally viewed in electron micrographs of untilted, negatively stained specimens, the junction lattice should appear mirror-symmetric in projection (with p6m plane group symmetry).Conventional high-irradiation micrographs of negatively stained specimens show doughnut-shaped connexons that appear nearly circularly symmetric in the Fourier-averaged images, and, therefore, the hexagonal lattice array, appear to have mirror symmetry (6). Higher resolution, hexagonally averaged reconstructions calculated by Zampighi and Unwin (33) from minimal irradiation micrographs of two forms of gap junctions, negatively stained with uranyl acetate, showed hexagonal shaped connexons arrayed with approximate mirror symmetry in the projected lattice images. However, the hexagonally shaped connexon images occured in ...

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The structure of the major protein constituent of photosynthetic membranes in higher plants, the chlorophyll a/b-light harvesting complex (LHC), was studied by x-ray diffraction and electron microscopy. The LHC was purified from Triton X-100 solubilized thylakoid membranes of the pea, and contained 6 mol of chlorophylls a and b per mole of a polypeptide of 27,000 molecular weight. X-ray diffraction showed that in the presence of 10 mM MgCl2, purified LHC forms planar aggregates that stack with a period of 51 A. Within each layer, LHC molecules pack with a center-to-center distance of 85 A but without long-range order. However, when LHC is incorporated into single-walled vesicles of plant lecithin, the addition of NaCl above 10 mM, or MgCl2 above 2 mM, led to the formation of plaques of hexagonal lattices, with a lattice constant of 125 A. The large domain size and high degree of order in the plane of the membrane are evident from the sharp lattice lines observed to 7 A resolution on the equator of the x-ray pattern. Freeze-fracture electron micrographs demonstrated an aligned stacking of the lattices in adjacent membranes, resulting in crystallinity in the third dimension over short distances. Micrographs of negatively stained membranes revealed a hexagonal lattice of the same lattice constant, formed by surface-exposed parts of the LHC molecules which are probably responsible for the ordered stacking of lattices. In both the LHC aggregates and in the reconstituted membrane lattices the diffracted x-ray intensities at 10-A spacing on the equator indicate that the LHC molecule contains paralled alpha-helices or beta-sheets that are oriented perpendicular to the planar arrays.

Multilamellar stacking seen in negatively stained lipid vesicle-tubulin mixtures has been attributed to lipid-protein interactions (Caron, J . M., and R. D . Berlin, 1979, 1. Cell Biol. 81 :665-671) . We show that this stacking is produced by the phosphotungstic acid used for staining, independent of the presence of tubulin in the sample . The morphology of negatively stained single bilayer vesicles obtained from dimyristoyl phosphatidylcholine or egg lecithin is specifically dependent upon the choice of metal stain. Uranyl oxalate maintains the appearance of unilamellar vesicles . After staining with sodium tungstate, the lipids form a network of multilayered lamellae with a periodicity of -55 Á. Phosphotungstic acid produces stacks of flattened vesicles with a period of -115 Á as well as broader multilamellar structures having a 55 Á repeat . The stain-determined morphology is not markedly altered by sample concentration, incubation time, or temperature, or by the presence of tubulin .Caron and Berlin (4) recently reported that tubulin and the high molecular weight proteins associated with microtubule assembly selectively adsorb to unilamellar dimyristoyl phosphatidylcholine liposomes. Negative staining of the proteinlipid mixtures with phosphotungstic acid (PTA) showed stacks of flattened liposomes and/or larger multilamellar structures resulting from fusion of the stacks .We have found that identical images of stacked vesicles and multilamellar structures can be produced with negatively stained liposomes that contain no tubulin . The appearance of these negatively stained lipid preparations is specifically dependent on the metal stain used, rather than on the presence of protein . The possibility of a morphology being induced by the stain must be critically evaluated in this and other negatively stained lipoprotein systems . MATERIALS AND METHODS Preparation of LipidsDimyristoyl phosphatidylcholine (DMPC) and egg lecithin were obtained from Sigma Chemical Co., St . Louis, Mo. Lipids were suspended in chloroform, dried overnight under vacuum to remove solvent, and swollen for 6-18 h in phosphate-buffered 0.1 N saline (pH 6.75; 2 mM EDTA). The multilayered liposomes (10 mg of lipid/0.6 ml of buffer) were sonicated in a bath sonicater at 26°-30°C until nearly optically clear. Preparation of TubulinOnce-and twice-cycled tubulin proteins were prepared from homogenates of gray matter from adult cattle, following the procedures of Caron and Berlin (4). THE JOURNAL OF CELL BIOLOGY " VOLUME 86 SEPTEMBER 1980 881-884 © The Rockefeller University Press " 0021-9525/80/09/0881/04 $1 .00 Lipid-Tubulin MixturesThe cold-soluble proteins resulting from one or two cycles of microtubule assembly were incubated with sonicated DMPC or egg lecithin vesicles at 30°C for at least 30 min. Lipid and protein were each suspended in 0.1 M NaCl, 0.02 M phosphate, pH 6.75, 2 mM EDTA, and mixed in ratios of 10 mg of lipid/0.5 mg of protein. Several specimens were prepared with higher protein content (10 mg of lipid/5 mg of protein...