The ribbons of the fusion peptides are colored green. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Voxels are colored according to their distances to the three-fold axis. (D to F) Surface-shadowed and ribbon diagrams showing the residue densities of the “zero out” maps for the three low pH conformations. (A) A representative raw micrograph (top) and 2D class averages (bottom) of the HA-Fab complex at pH 7.8. Fusion HA! Related GIFs. The bound Fab F005-126 has direct interactions with residues 171–173, 239, 240 of one HA protomer and residues 91–92, 270–273, 284–285 of a neighboring protomer (S5 Fig). Images of the HA-Fab complex at neutral pH and at low pH were recorded using a Titan Krios electron microscope (Thermo Fisher) operating at an acceleration voltage of 300 keV and equipped with a GIF Quantum energy filter (slit width 20 eV) and a Gatan K2 Summit camera. Histograms indicate the portion of voxels with a particular resolution. Hairpin 1 (21–38), orange. GIF. The fitting was done in Chimera by treating the beta sheet and the C-terminal helices of the stem region as a rigid body and by maximizing the density values around the fitted atoms [52]. Residues in blue belong to Helix A. The contacting interface was calculated with Chimera. The amount of HA loaded in each lane was adjusted to be the same. However, the HA-Fab-pH 5.2 conformations B and C showed significant differences in the stem region from those of the HA-Fab-pH 7.8 and the HA-Fab-pH 5.2 conformation A (Figs 2, S9 and S10). Yes We then determined the cryo-EM structures of the full-length HA in complex with Fab F005-126 at pH 7.8 and pH 5.2. (C) Models of pH 5.2 conformation C (hot pink) and pH 5.2 conformation A (gray) are superimposed by using the head domains. HA1 subunit (9–324), gray. The central helices of the HA-Fab-pH 5.2 conformation C adopt a straight and extended conformation rather than the bent form observed in other conformations (Fig 3A). Middle, pH 5.2 conformation B. Are you ready to find out your quirk? Bidoof's black paws have three toes with some webbing between them. Right: Zoomed-in views showing the Helix As in neutral (red) and low pH (yellow) conditions. The steric clashes between the models were calculated with Chimera (Fig 4). Two atoms are considered to have close contacts if the distance between them minus the sum of their van der Waals radii is less than 1. https://doi.org/10.1371/journal.ppat.1009062.s005. Unlike the canonical coiled coil, the C-terminal Helix Ds are loosely packed and do not have a hydrophobic core. 1021 GIFs. Search, discover and share your favorite Vegeta GIFs. Bidoof is a brown, rodent-like Pokémon with four short legs. For more information about PLOS Subject Areas, click Then, two parallel rounds of 3D classification were conducted, yielding six classes and five classes, respectively. Residues not conserved are colored white. Peak fractions containing both HA and Fab F005-126 were collected and concentrated to ~ 0.4 mg/ml for cryo-EM sample preparation. Let's go. Helix A (39–56), blue. Structure superimpositions of the pH 5.2 conformation A (left, green), pH 5.2 conformation B (middle, gold) and pH 5.2 conformation C (right, hot pink) with the conformation at pH 7.8 (gray), respectively. Particles from two classes showing conformational changes in the central helixes were combined and subjected to final 3D refinements and reconstructions, yielding a 3.4 Å density map (pH 5.2 conformation C). The surface is colored according to the surface electrostatic potential with positive charges colored blue and negative charges colored red. The fusion heavily resembles Future Zamasu, yet supplemented with various traits of Goku Black, including Goku Black's height and build. The cryo-EM structure of the HA at pH 7.8 is highly similar to the previously determined crystal structure of the HA ectodomain from an H3N2 virus (PDB accession number: 3WHE) [24]. Miembro del Clan Grupo: Miembros Mensajes: 223 Agradecimientos: 309 Registrado: 12-July 11 Desde: santa marta Miembro No. The hemagglutinin (HA) glycoproteins of influenza viruses play a key role in binding host cell receptors and in mediating virus-host cell membrane fusion during virus infection. One of these classes showed similar overall features to those of the pH 7.8 conformation except for the fusion peptides. (B) Local resolution map. Scale bars in the raw micrographs represent 50 nm. No, Is the Subject Area "Viral structure" applicable to this article? These results provide new insights into the structural transition of HA during virus entry. In addition, the conformational changes of the central helices completely alter the properties of the fusion peptide binding pockets and the inner and outer surface of the central helices (Figs 3C, 3D and S13). The stem specific antibody 31.a.83 at a concentration of 10 μg/ml was serially diluted and added to the microplate well. Conceptualization, The N-terminal hydrophobic residues of the fusion peptide are shown as orange spheres. After one round of 3D classification, 486,746 particles were selected and subjected to 3D refinement. The fusion's hairstyle looks mostly like Goku Black's as Super S… Visualization, See Materials and methods for details. However, the coiled-coil parameters of the superhelical radius and superhelical frequency calculated for the Helix Ds of the pH 7.8 conformation are 9.2 Å and -3.0° per residue, respectively, whereas those for the Helix Ds of the HA-Fab-pH 5.2 conformation C are 10.1 Å and -0.8° per residue, respectively (Fig 3A). The stem specific antibody 31.a.83 in full length was used in the ELISA assay instead of its Fab part. The structure models were built and adjusted in COOT [45] and were refined by using PHENIX [46] real-space refinement with secondary structure and geometry restraints. Left: Ribbon diagrams showing the position of Helix A in the stem region. All the maps were low-pass filtered to 8.3 Å. Competing interests: The authors have declared that no competing interests exist. #freddy Funding: This work was supported by funds from the Ministry of Science and Technology of China (grant: 2016YFA0501100), the National Natural Science Foundation of China (grants: 31925023, 21827810, 31861143027 & 31470721), the Beijing Frontier Research Center for Biological Structure and the Beijing Advanced Innovation Center for Structure Biology to Y.X. The Helix Ds are more open and have a larger twist compared to the simulated coiled coil. We have now used cryo-EM to determine the structures of full-length hemagglutinin solubilized in detergent, alone, and in complex with the Fab of an infectivity neutralizing antibody. The hydrophobic distal ends of the fusion peptides insert deeply into the pockets between the Helix Ds and form a hydrophobic core of residues Leu2 and Phe3 (Fig 1E and 1F). The “zero out” maps were calculated by setting the values of the map grid points within a radius of 2.5 Å of each fitted model atom to zero [52]. (A-D) Representative 2D class averages (A), local resolution (B), particle orientation distribution (C) and directional FSC plots of the HA-Fab reconstructions at pH 5.2 (D). pathogens and how they interact with host organisms. The final pH was verified with a pH meter. In addition, wrapping by the HA1 head should further stabilize the membrane-distal Helix Cs. The stem specific antibody 31.a.83 was serially diluted and detected for its binding to HA. (A) Sequence alignments of the HA2s. Particles from classes with similar features to those of the HA-Fab conformation at pH 7.8 were combined and subjected to final 3D refinements and reconstructions, yielding a 3.0 Å density map (pH 5.2 conformation A). No, Is the Subject Area "Serine proteases" applicable to this article? Side view (left) and bottom-up view (right) are shown. Residues completely conserved in both the group 1 and group 2 HA2s are colored black. The conformational changes enlarged the distances between the membrane-proximal ends of the central helices and created an inner cavity with a volume of approximately 1400 Å3 (S12 Fig). Residues completely conserved in the group 2 HA2s are colored black. With Tenor, maker of GIF Keyboard, add popular Ha animated GIFs to your conversations. MolProbity [47] and EMRinger [48] were used to evaluate the final refined models. He retains the same appearance in Dragon Ball Z: Dokkan Battle, however, while his card ap… Atomic models of the HA ectodomain and the bound Fabs were built (Fig 1B and 1C). However, a universal vaccine is still lacking and drug resistance is an emerging problem due to the fast mutation and evolution of influenza virus. The three HA1/HA2 heterodimers are colored hot pink, cornflower blue and orange, respectively. The recombinant HA was eluted with the balancing buffer containing 5 mM d-desthiobiotin (Sigma, D1411). Subsequent 2D and 3D analyses were performed using RELION [42]. Different conformations were observed. The epitopes recognized by F005-126 at pH 7.8 and at pH 5.2 are the same and are highly similar to these observed in the crystal structure of HA and Fab F005-126 (S5 Fig). The bound Fab F005-126 prevents the pre-postfusion transition of the HA and renders the HA trypsin resistant. No, Is the Subject Area "Crystal structure" applicable to this article? The bound antibody was eluted with 0.1 M glycine at pH 3.5. Resources, The three central helices of the HA trimer intertwine to form a left-handed triple-stranded coiled coil (Fig 1D). The fusion peptide and the nearby stem region of HA could undergo initial conformational changes that further prime subsequent conformational changes, disassociation of the HA1 head and structure rearrangement of the HA2 stem [13,15,16]. https://doi.org/10.1371/journal.ppat.1009062.s014. https://doi.org/10.1371/journal.ppat.1009062.g005. The transfected cells were harvested 48 hours posttransfection by centrifugation at 1000×g for 5 minutes. Based on these observations, it should be possible to trap the pre-postfusion transition of the HA trimer in possible intermediate states by stabilizing the HA1 head. (A) Structure comparisons of the Helix As in neutral and low pH conformations. After concentration and buffer exchange, the supernatant was loaded onto a protein A Sepharose column (GE Healthcare, 17-0780-01). The backbone of the fusion peptide is shown in green. He possesses Future Zamasu's green skin, gray irises, and white hair; however, his face has more of Goku Black's jawline. Moreover, our research also suggests that release of the fusion peptide and unwinding of the central helices are likely to be independent to the dilation of the HA head which is a prerequisite for subsequent conformational change including the extending of HA2. The head and central helices are shown in gray. The head encompasses the sialic acid receptor binding domains (RBDs), while the stem has a hydrophobic fusion peptide at the N-terminus and the transmembrane helix at the C-terminus of each HA2. The profiles of the disordered transmembrane domain and the bound detergent micelle (map contoured at 3 σ) are indicated by brown lines. Side view (left) and bottom view (right) are shown. The major epitopes recognized by several characterized broadly neutralizing antibodies of HA are around Helix A of HA2 in the stem region [20,25,29–32]. After one round of 3D classification, 359,922 particles were selected and subjected to 3D refinement. (A) A schematic diagram showing the domain organization of the influenza virus HA. The antibody was cloned into a specialized vector with a preinserted region encoding a signal peptide (GWSCIILFLVATATGVHS) and a human IgG1 antibody constant region (Fc) [37]. Particles were automatically selected using Gautomatch [41]. (B-C) The contacting interface (in red) of HA with Fab F005-126 in different conformations. HA head could be a promising target for neutralizing antibodies not only by blocking interaction with receptor but also by preventing the low pH induced structure transition. Particles from this class were selected and subjected to final 3D refinements and reconstructions, yielding a 4.2 Å density map (pH 5.2 conformation B). (A-D) Densities around the central helix (residues 76–125 of HA2) and the beta sheet (residues 9–18 of HA1, 21–38 and 126–141 of HA2) in the stem region of different conformations.
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