Journal: Trends in immunology

Article Title: The many ways in which alphaviruses bind to cells

doi: 10.1016/j.it.2023.11.006

Figure Lengend Snippet: Cryo-EM reconstructions of CHIKV VLPs in complex with mouse MXRA8 ( left , receptor in magenta), VEEV VLPs in complex with LDLRAD3 LA1 ( middle , receptor in yellow), and SFV VLPs in complex with VLDLR-LBD ( right , receptor in green) adapted from EMD-9394, EMD-24116, and EMD-35451 (Electron Microscopy Databank, https://www.ebi.ac.uk/emdb ), respectively. Atomic models are shown in the bottom part, with receptors displayed as ribbons and viral proteins as surface renderings, with E1 colored tan, E2 A domain colored green, E2 B domain colored blue, and the remainder of E2 shown in pale purple. Receptors are colored magenta/pink (MXRA8), yellow (LDLRAD3), or green (VLDLR). Models are adapted from PDB: 6NK6, PDB: 7N1H, and PDB: 8IHP ( https://www.rcsb.org ), respectively. All structures were visualized in ChimeraX [ 40 ].

Article Snippet: Cryo-EM reconstructions of CHIKV VLPs in complex with mouse MXRA8 ( left , receptor in magenta), VEEV VLPs in complex with LDLRAD3 LA1 ( middle , receptor in yellow), and SFV VLPs in complex with VLDLR-LBD ( right , receptor in green) adapted from EMD-9394, EMD-24116, and EMD-35451 (Electron Microscopy Databank, https://www.ebi.ac.uk/emdb ), respectively.

Techniques: Cryo-EM Sample Prep, Electron Microscopy

Journal: Science Advances

Article Title: In situ structure of the human gap junction

doi: 10.1126/sciadv.aea4183

Figure Lengend Snippet: ( 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), 80 S 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, 80 S 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 80 S ribosomes and microtubule segments (MTs) to the Cx43 GJCs, calculated from multiple cryo-ET reconstructions ( n = 26). The 15-nm-wide zone occupied by Cx43 channels is indicated in green. The 22-nm-wide ribosome exclusion zone (REZ) is indicated in gray.

Article Snippet: The cryo-ET map of the Cx43 lattice has been deposited in the Electron Microscopy DataBank (EMDB) with the deposition ID EMD-54024.

Techniques: Labeling, Tomography, Clinical Proteomics

Journal: Science Advances

Article Title: In situ structure of the human gap junction

doi: 10.1126/sciadv.aea4183

Figure Lengend Snippet: ( 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 the top (+ to – direction) for the extracellular region (gap), inner leaflet (IL1), and intracellular densities (OL1). ( I ) Rendering of the CG MD simulation setup. The connexins are green, the POPC lipids are blue (head groups in a darker shade), and cholesterol is magenta. The solvent water is rendered as a transparent cube. The inset shows the area indicated. ( J and K ) Number of POPC lipids (J) and cholesterol molecules (K) around the centermost gap junction hemichannel is plotted as a function of the simulation time for both the membranes (+ and – sides).

Article Snippet: The cryo-ET map of the Cx43 lattice has been deposited in the Electron Microscopy DataBank (EMDB) with the deposition ID EMD-54024.

Techniques: Labeling, Solvent

Journal: Science Advances

Article Title: In situ structure of the human gap junction

doi: 10.1126/sciadv.aea4183

Figure Lengend Snippet: ( 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 density below it (stem) and the density above it [lateral contacts layer (LCL)].

Article Snippet: The cryo-ET map of the Cx43 lattice has been deposited in the Electron Microscopy DataBank (EMDB) with the deposition ID EMD-54024.

Techniques: Tomography, Single Particle, Cryo-EM Sample Prep, Comparison, Membrane

Journal: Nature Communications

Article Title: CryoEM and computational modeling structural insights into the pH regulator NBCn1

doi: 10.1038/s41467-025-64868-z

Figure Lengend Snippet: a CryoEM density map of the NBCn1 dimer with the monomers colored in violet and pink. b Atomic model of the NBCn1 dimer in the same orientation as in ( a ). TMs 1-14 and helices H1-10 are shown as cylinders. The N-glycosylations (NAG) are shown in green and labeled accordingly. c Topology and domain arrangement of the NBCn1 monomer. Four highly conserved cysteines are shown as red dots. The branched structures at Asn776, Asn786, and Asn796 represent N-linked glycosylation. d Ribbon diagram of the atomic structure of an NBCn1 monomer with the gate (violet) and core (pink) domains. Side (left) and top (right) views are shown. The regions involved in the formation of the two disulfide bonds are highlighted in yellow. e CryoEM densities corresponding to ions are shown in purple. The atomic model is overlaid with the cryoEM map. f CryoEM densities corresponding to N-glycosylations at Asn776, Asn786 and Asn796 are shown. The atomic model is overlaid with the cryoEM map.

Article Snippet: The final cryoEM density map of NBCn1 has been deposited to the Electron Microscopy DataBank (EMDB) under the accession code EMD-70906 (the main cryoEM density map).

Techniques: Labeling, Glycoproteomics

Journal: Nature Communications

Article Title: CryoEM and computational modeling structural insights into the pH regulator NBCn1

doi: 10.1038/s41467-025-64868-z

Figure Lengend Snippet: a Surface-rendered NBCn1 monomer model showing an extracellular-facing cavity. Na + and CO 3 2 − ions are shown as purple spheres and green-red sticks, respectively. b Magnified view of the cryoEM densities of Na + , CO 3 2 − and surrounding residues at the same contour level of 0.34σ. c The Na + and CO 3 2 − ions coordinated at TM1, TM3, TM5, TM8 and TM10 in S1 cryoEM (OF) and S2 cryoEM (OF) sites.

Article Snippet: The final cryoEM density map of NBCn1 has been deposited to the Electron Microscopy DataBank (EMDB) under the accession code EMD-70906 (the main cryoEM density map).

Techniques:

Journal: Nature Communications

Article Title: CryoEM and computational modeling structural insights into the pH regulator NBCn1

doi: 10.1038/s41467-025-64868-z

Figure Lengend Snippet: The long EL3 is omitted for clarity. a SILCS maps of OF and IF NBCn1. Cation and anion densities are presented as purple and green mesh respectively. The acidic and basic residues lining the OF and IF cavities are shown as red and blue sticks, respectively. The central residue D930 is shown as red spheres. b Average maps from 1 µs MD simulations. Na + and CO 3 2 − densities are presented as purple and green mesh respectively. The acidic and basic residues lining the OF and IF cavities are shown as red and blue sticks, respectively. The central residue D930 is shown as red spheres. c OF (left) and IF (middle) state with their corresponding cumulative CO 3 2 − densities (green mesh) calculated from series of MD simulations with position restraints on the C-atom of CO 3 2 − , which prevents it from moving along the z-axis. The coordinate system used in the simulations is also shown. Free energy map (right) projected in the xz-plane and corresponding to the CO 3 2 − densities in the OF and IF state. Four putative binding sites are designated on the map from the four minima closest to the center of the protein. The ~5 Å vertical displacement of the S1 MD site is shown as well. d Amino acid composition of the four binding sites identified in ( c ). The color coding is as follows: Gate/gate residues (purple helices/sticks), core/core residues (pink helices/sticks in the OF state and yellow helices/sticks in the IF state), CO 3 2 − (green sticks), Na + purple spheres.

Article Snippet: The final cryoEM density map of NBCn1 has been deposited to the Electron Microscopy DataBank (EMDB) under the accession code EMD-70906 (the main cryoEM density map).

Techniques: Residue, Binding Assay

Journal: Nature Communications

Article Title: CryoEM and computational modeling structural insights into the pH regulator NBCn1

doi: 10.1038/s41467-025-64868-z

Figure Lengend Snippet: The data is depicted as percent of wt-NBCn1 function divided by percent of wt-NBCn1 cell-surface PM expression. NBCn1 wt ( n = 18 biologically independent experiments) and single cysteine functional mutant data: F614C ( n = 5, p < 0.0001); C619A ( n = 8, p < 0.0001); T625C ( n = 3, p < 0.0001); L665C ( n = 16, p < 0.0001); G666C ( n = 12, p < 0.0001); S667C ( n = 7, p < 0.0001); T668C ( n = 12, p < 0.0001); G669C ( n = 4, p = 0.4859); P670C ( n = 8, p = 0.2164); T720C ( n = 7, p < 0.0001); E724C ( n = 4, p < 0.0001); F727C ( n = 4, p < 0.0001); I731C ( n = 6, p < 0.0001); I734C ( n = 4, p = 0.0001); E738C ( n = 7, p = 0.0487); E741C ( n = 6, p < 0.0001); K742C ( n = 3, p < 0.0001); D745C ( n = 4, p < 0.0001); E748C ( n = 8, p < 0.0001); K843C ( n = 6, p < 0.0001); Q844C ( n = 3, p = 0.9992); K846C ( n = 11, p < 0.0001); S860C ( n = 10, p = 0.0001); D861C ( n = 6, p < 0.0001); D930C ( n = 8, p < 0.0001); Q931C ( n = 3, p < 0.0001); I933C ( n = 7, p < 0.0001); T934C ( n = 4, p < 0.0001); V974C ( n = 4, p < 0.0001); A975C ( n = 7, p < 0.0001); A976C ( n = 4, p < 0.0001); T977C ( n = 5, p < 0.0001); V978C ( n = 8, p < 0.0001); L979C ( n = 9, p = 0.0005); S980C ( n = 10, p = 0.0003); S1047C ( n = 5, p < 0.0001); K1050C ( n = 7, p = 0.0379); K1100C ( n = 3, p = 0.8068); P1109C ( n = 4, p < 0.0001); D1135C ( n = 4, p = 1.0000); D1136C ( n = 7, p < 0.0001); and L1137C ( n = 4, p < 0.0001). One-way ANOVA and Dunnett’s test were used to compare multiple study group means with wt-NBCn1. Statistically significant results differing from wt-NBCn1 are depicted as mean ± SEM (# p < 0.05, + p < 0.01 and * p < 0.0001). Source data are provided as a Source Data file. Residues are numbered based on NBCn1-A for structural comparison.

Article Snippet: The final cryoEM density map of NBCn1 has been deposited to the Electron Microscopy DataBank (EMDB) under the accession code EMD-70906 (the main cryoEM density map).

Techniques: Expressing, Functional Assay, Mutagenesis, Comparison