In situ structure of the human gap junction

Figures

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  Fig. 1.. Cryo-ET and template matching of HEK293 Cx43-eGFP cells. (A) A schematic representation of the gap junction assembly from connexin 43 (Cx) monomers to hemichannels and to the gap junction plaque between two cells. Intra- (IL) and extracellular (EL) loops are labeled. The transmembrane (TM) helices are numbered 1 to 4. (B) One-nm-thick slice through a cryo-ET reconstruction of a cell-cell junction. Areas harboring Cx43 channels (green), 80S ribosomes (cyan), and microtubules (pink) have been indicated. A mitochondrion has been labeled (M). Scale bars, 100 nm. (C) Close-up of the area indicated in (B). Side views of Cx43 GJCs bridging two plasma membranes are indicated with arrowheads. Scale bar, 10 nm. (D) Close-up from a different cryo-ET reconstruction showing top views of the Cx43 GJCs. Scale bar, 10 nm. (E) Results of template matching for Cx43 GJCs, 80S ribosomes, and microtubule segments are shown after plotting the templates back onto the cryo-ET tomogram (illustrated with a 1-nm-thick slice in the background). The gap junction is depicted as a transparent surface (green) fitted to the Cx43 channel positions for visual clarity. (F) Histogram of pairwise distances between…

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  Fig. 2.. Subtomogram averaging of Cx43 GJCs. (A) Slice through a subtomogram average of the Cx43 lattice is shown. One channel is indicated (arrowhead). Scale bar, 10 nm for (A) to (C). (B) Slice through the subtomogram average, along the solid line in (A). (C) Slice along the dashed line in (A). Channels are indicated in (B) and (C) (arrowheads). (D) Isosurface of a subtomogram average. A low-pass filter to 20-Å resolution has been applied to improve the interpretability of the lateral layers. Cx43 atomic models (PDB: 7Z22, residues 17 to 105 and 151 to 235) are shown in green. Two hemichannels forming a full channel are indicated (arrowheads). The convex (+) and concave (−) sides of the lattice are indicated in (B) to (D). (E) Density distribution as a function of distance from the lattice midpoint is shown. Density is in arbitrary units. The inner (IL) and outer (OL) leaflets of the two lipid bilayers are marked. The regions corresponding to intracellular density bridging Cx43 hexamers, denoted as lateral-contact layers (LCLs), are labeled. An additional layer of density on the concave side of the gap junction is indicated with an asterisk. (F to H) Isosurface renderings are shown from…

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  Fig. 3.. Structure of the Cx43 channel. (A to C) Isosurface renderings of the cryo-ET subtomogram average at 14-Å resolution segmented with a cylindrical mask are shown as gray transparent surfaces, together with a partial atomic model of the Cx43 channel (PDB: 7Z22, residues 17 to 105 and 151 to 235). The inset indicates the level of the cross sections taken from the middle of the top bilayer (A), the middle of the extracellular loops (B), and the middle of the bottom bilayer (C). The TM helices are numbered 1 to 4 for one Cx monomer in both hemichannels. The convex (+) and concave (−) sides of the gap junction are indicated. (D) Close-up of the hemichannel cryo-ET map (this study) and a single-particle cryo-EM structure of the Cx43 hemichannel (EMD-14475), low-pass filtered to the same resolution (14 Å) for comparison. At this resolution, the N-terminal helices (residues 1 to 16, purple) closing the channel are visible in the cryo-EM density. The dashed lines indicate the position of the two membrane leaflets in both structures. (E and F) Close-ups of the intracellular areas indicated in the inset (dashed rectangles) are shown. In both, a stalk-like density can be seen, connecting the…