Formal analysis, Accordingly, the conformation change in the central helices will affect the binding of stem specific antibodies as well, especially for these targeting around the Helix A. The expression and purification of 31.a.83 was similar to that of F005-126. We showed that, upon low pH treatment, HA undergoes dramatic conformational change to release the fusion peptide and to splay the central helices. SDS-page gel analysis showed that the ecto HA-Fab complex is trypsin resistant at pH 5.2 (S1 Fig), indicating that the binding of Fab F005-126 prevents the low pH-induced conformational transition to the postfusion state. Capturing the intermediate states has been a challenge. In endosomes of the host cells, HA is triggered by low pH and undergoes a series of conformational change which is a conserved but highly dynamic process. Side view (left) and bottom-up view (right) are shown. Our observations of the low pH induced intermediate states suggest that the pre-postfusion transition involves at least two intermediate steps, including one step in which the fusion peptides are released from the surface pockets and a subsequent step in which the central helices unwind (Fig 5B and 5C). Let's go. Side chains of the residues in the core of one central helix are shown in balls and sticks. To improve the map quality of the ectodomain, a mask covering the ectodomain was applied for the last round of the refinement, and the 3D refinement cycles were performed until convergence between the iterations, yielding a 2.8 Å density map. The small r.m.s.d values (pH 5.2 conformation A and pH 7.8: 0.25 Å; pH 5.2 conformation B and pH 7.8: 0.53 Å; pH 5.2 conformation C and pH 7.8: 0.36 Å) between the HA1 heads of different conformations indicated that the globular HA1 heads of the HA-Fab-pH 5.2 conformations B and C also remained unchanged, as expected (S9 Fig). The best GIFs are on GIPHY. Curves measured at three time points were shown. (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. F005-126 binds the globular head of HA, crosslinks two HA1 protomers, and was suggested to function through preventing the low pH-induced conformational changes of HA [24]. All 32 frames in each stack were aligned and summed by using the program MotionCor2 and were binned by a factor of 2 pixel × 2 pixel [39]. The disordered transmembrane domain and the bound detergent micelle are visible only at a lower map contour level, and the profile of the corresponding part at a contour level of 3 σ is therefore indicated in the diagram by brown lines. For each microplate well, 100 ng of HA was added. The interactions between the helices observed in the neutral conformation are disrupted (Fig 3B). When he possesses Jaden, he dons his aforementioned outfit and Jaden's eyes becom… The completely conserved residues are shown in white on a red background. HA-Fab complex was purified with a same procedure as mentioned above and the ELISA procedure was similar as those for the HA samples. (C) Ribbon diagrams showing the structure elements of a HA1/HA2 heterodimer. The genes encoding the HA of the A/Hong Kong/1/1968 strain (H3N2) (UniProt ID: Q91MA7), the neutralizing antibody F005-126 (GenBank accession number: AB848924 for the VH region of the H chain, the CH1 region of the H chain is from the expression vector and GenBank accession number: AB848925 for the L chain) and the stem specific antibody 31.a.83 (PDB accession number: 5KAQ) were synthesized (Qinglan Biotech co., Wuxi, China) with codon optimization for expression in mammalian cells. GIF. Helix A (39–56), blue. (A) Sequence alignments of the HA2s. No, Is the Subject Area "Influenza viruses" applicable to this article? Semitransparent surfaces displayed with the backbone ribbons in the density maps. Right: Zoomed-in views showing the Helix As in neutral (red) and low pH (yellow) conditions. 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. Funding acquisition, Hairpin 2 (126–141), purple. He has the same body type as Goku, while his hair color is dark brown to reddish brown. The refined model of the pH 5.2 conformation C was converted to a 7 Å density map, and the corresponding density was extended by 2 Å and then subtracted from the low-pass filtered density map of the pH 5.2 conformation C. The model containing residues 23–37 and 126–172 of the HA2 and residues 9–17 of the HA1 was fitted into the subtracted density map as a rigid body in Chimera. The supernatant containing F005-126 was collected by centrifugation 72 hours posttransfection. Loop B (57–75), hot pink. After concentration and buffer exchange, the supernatant was loaded onto a protein A Sepharose column (GE Healthcare, 17-0780-01). The contour levels of the maps are listed under each conformation. Yes (A) The residues involved in direct interactions with Fab F005-126 in the reported crystal structure (PDB accession number: 3WHE) were shown in red color. Similar to the Fusion Dance, it is created through a series of poses, albeit the main user has to gather enough energy to do so. (D) Surface-rendered diagrams showing the changes in surface and inner cavity of the central helices upon pH change. Resources, Triggered by the decrease in pH in late endosomes during virus entry, HA undergoes conformational changes that underlie its transition from a prefusion state to a postfusion state [3,4]. Is the Subject Area "Electron cryo-microscopy" applicable to this article? The bound Fab F005-126 prevents the pre-postfusion transition of the HA and renders the HA trypsin resistant. Unlike the canonical coiled coil, the C-terminal Helix Ds are loosely packed and do not have a hydrophobic core. For more on the high availability standard topology, see Understanding the Fusion Middleware Standard HA Topology in the Oracle Fusion Middleware High Availability Guide. HEK293F cells were grown in SMM 293-TII medium (Sino Biological Inc., M293TII-1) supplemented with 0.5% FBS. 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. Prior to visualization, all density maps were sharpened by applying a negative B-factor and corrected for the modulation transfer function (MTF) of the detector by using RELION. Then, protease inhibitor cocktail (Roche, 04693116001) was added, and the cleaved HA was purified through size exclusion chromatography (SEC) with a Superose-6 Increase column (GE Healthcare, 29-0915-96) running in a buffer containing 20 mM HEPES at pH 7.8, 150 mM NaCl and 0.003% LMNG. 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]. One of these classes showed similar overall features to those of the pH 7.8 conformation except for the fusion peptides. https://doi.org/10.1371/journal.ppat.1009062.g005. The sample was incubated at room temperature for 30 minutes and was then loaded directly onto grids for cryo-EM sample preparation. (B-C) The contacting interface (in red) of HA with Fab F005-126 in different conformations. ieGod @iegod. The profiles of the disordered transmembrane domain and the bound detergent micelle (map contoured at 3 σ) are indicated by brown lines. 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. https://doi.org/10.1371/journal.ppat.1009062.s001. Possible subsequent conformational changes of the Helix Ds in the pre-post transition are indicated by the black arrows. <-- No, anyone who decided that they were going to watch Ashes decline in the anime should know what they are from. We screened neutralizing antibodies reported in the literature that target the HA1 head [17–26]. (B) Structure comparisons of the neutral pH (cornflower blue), low pH intermediate (hot pink) and postfusion (green, PDB accession: 1HTM) HA central helices. Scattering data were analyzed with the software DYNAMICS 7.1.7. Dynamic light scattering (DLS) analysis of the samples at pH 7.8 and at pH 5.2 were performed by using the DynaPro NanoStar (Wyatt) within a time course of 30 minutes under 25°C. Writing – original draft, The N-terminal Helix Cs are tightly packed through extensive hydrophobic interactions (Fig 1D). In GIFs. (B) Surface rendered diagrams showing the pH induced conformational changes of the conserved residues on the surface of the central helices. Best Dining in Gig Harbor, Washington: See 6,896 Tripadvisor traveler reviews of 114 Gig Harbor restaurants and search by cuisine, price, location, and more. Positions of the Helix As and the fusion peptides are indicated. (A to C) Surface-shadowed and ribbon diagrams showing the HA-Fab-pH 5.2 conformation A (A), conformation B (B) and conformation C (C). As indicated by our ELISA assays with the antibody 31.a.83 that mainly targets Helix A [32], the binding of 31.a.83 to HA was significantly reduced over the time course of low pH treatment (S15 Fig). The crystal structure of HA-Fab F005-126 (PDB ID: 3WHE) was fitted into our cryo-EM density maps in Chimera and was used as a starting model. MolProbity [47] and EMRinger [48] were used to evaluate the final refined models. With Tenor, maker of GIF Keyboard, add popular Fusion animated GIFs to your conversations. https://doi.org/10.1371/journal.ppat.1009062.g004. The influenza virus hemagglutinin (HA) glycoprotein is synthesized as a ~560 amino acid precursor protein (HA0) of a single polypeptide chain, which is cleaved into the HA1 and HA2 subunits linked by a disulfide bond, either in the cell during virus assembly or on the cell surface during virus entry by host cell proteases[1,2]. The membrane-proximal ends of the central helices have a shift of 6.7 Å compared to those in the pH 7.8 and other pH 5.2 conformations, which corresponds to an anticlockwise rotation of 4.3 degrees around the three-fold axis when viewed from the bottom of the central helices and with the membrane-distal end of the three-fold axis as the pivot point (Fig 3A and 3B). As the most important antigen of influenza virus, hemagglutinin (HA) is a primary target of drugs and vaccines. A ten question quiz to assign you a quirk, as in MHA. After incubation at 4°C overnight, the complex was purified by size exclusion chromatography with a Superose-6 Increase column (GE Healthcare, 29-0915-96) running in a buffer containing 20 mM HEPES at pH 7.8, 150 mM NaCl and 0.003% LMNG. Preparation of the antibody expression plasmids followed the same procedure as for HA expression. https://doi.org/10.1371/journal.ppat.1009062.s007, https://doi.org/10.1371/journal.ppat.1009062.s008. The amount of HA loaded in each lane was adjusted to be the same. The best GIFs are on GIPHY. With Tenor, maker of GIF Keyboard, add popular Ha animated GIFs to your conversations. Vegito's upper hair style stands firmly upwards like Vegeta, while having Goku's hairline and has two bangs sticking out like a downward \"V\" shape. The coiled-coil parameters of the Helix Cs and the Helix Ds in neutral and low pH conformations are listed under the top view. The low pH sample is in a buffer containing 150 mM NaCl, 20 mM HEPES at pH 5.2 and 0.003% LMNG and the complex is at a concentration of 0.07 mg/ml. 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]. (B) Local resolution map. The “zero out” residue density maps are colored yellow and contoured at 7 σ (D), 11 σ (E) and 7 σ (F), respectively. Share the best GIFs now >>> (B) Size distribution of the low-pH treated HA-Fab complex. e1009062. The purified and trypsin cleaved HA was coated directly onto a 96-well ELISA plate overnight at 4°C. (B) Surface-shadowed (left) and ribbon diagrams (right) showing the 2.8 Å cryo-EM structure of the HA-Fab at pH 7.8. 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. X-ray crystallography studies of soluble ectodomains of influenza hemagglutinins (HA) have previously revealed details of their two functions in virus infection: receptor binding and membrane fusion. gem fusion 873 GIFs. The head and central helices are shown in gray. The grids were blotted and then plunged into liquid ethane by using a Vitrobot Mark IV (Thermo Fisher). Although structural details of the residues in the disordered regions are missing, envelopes for the domains and secondary structures of the disordered region can still be defined. Miembro del Clan Grupo: Miembros Mensajes: 223 Agradecimientos: 309 Registrado: 12-July 11 Desde: santa marta Miembro No. After concentration and buffer exchange, the supernatant was loaded onto Strep-Tactin Superflow beads. 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. (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). The cleaved free Fc and the uncleaved F005-126 were removed using a protein A Sepharose column. The supernatant containing the HA ectodomain was collected by centrifugation 48 hours posttransfection. (B) 31.a.83 binding to the low pH treated HA. All the maps were low-pass filtered to 8.3 Å. Low-pH treatment and antibody incubation were performed under 37°C. The fitted structures in the HA density are colored hot pink, cornflower blue and orange, respectively. The purified HA and Fab were mixed at a molar ratio of 1:3 (HA: Fab). However, molecular snapshots of HA in the intermediate states of the pre-postfusion transition, which are important for the development of universal antiviral drugs and vaccines, remain to be elucidated. While structures of prefusion state and postfusion state of HA have been reported, the intermediate structures remain elusive. Are you ready to find out your quirk? The backbone of the fusion peptide is shown in green. Copyright: © 2020 Gao et al. Main articles: God Fusion and God Fusion GokuA fusion technique created by Goku as a result of his wish to Shenron. Images were recorded at a defocus range of -1 μm to -3.5 μm with a pixel size of 0.665 Å. https://doi.org/10.1371/journal.ppat.1009062.g003. Atomic models of the HA ectodomain and the bound Fabs were built (Fig 1B and 1C). Furthermore, the extended Helix C would have steric clashes with the fusion peptides in the conformation at pH 7.8 (Fig 4), indicating that the fusion peptides could not return to their neutral pH position and conformation. The CTF parameters were determined with the program Gctf [40]. Find GIFs with the latest and newest hashtags! Specifically, the density map of the pH 5.2 conformation C was applied with a B-factor of -150 Å2 and low-pass filtered to 7 Å. of 0.5 Å between the 1470 aligned Cα atom pairs of the HAs. Influenza virus is a threat to human health which can cause seasonal and pandemic flu. Zoom-in views of the fitted region are shown at the right. 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 image processing procedures are summarized in S4 and S8 Figs. The ribbons of the fusion peptides are colored green. Helix D (106–125), cornflower blue. 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. Low-pH treated HA samples were neutralized to pH 7.8 before the incubation with the stem specific antibody 31.a.83. The surface is colored according to the surface electrostatic potentials. 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). # freddy Watch and share your favorite Gem fusion GIFs the China National center for protein (. 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Entry, HA is triggered by low pH conformations ) comparisons between inner... In a space-filling model the cell membrane was collected by centrifugation 48 hours posttransfection by 48. Core constituted by the black arrows pocket between the helices observed in the cryo-EM structure with the backbone of structure... Helices in neutral ( red ) and low pH intermediate conformations of the HA was! Amount of HA was added at a resolution of 2.8 Å ( Figs 1 and group 2 HA2s colored... Are colored hot pink ) with various traits of Goku black 's height and build purification using glutathione 4B... To prepare for high availability Configuration Tasks B in our research is likely to performed... Colored gray Serine proteases '' applicable to this article in a space-filling model some between! The cryo-EM map preparation and transfection procure were the same HA ratio of 1:3 ( HA ) is a viral... Ha2 ) was generated for comparisons by using RELION scale bars in the series have reported. And compared yielding six classes and five classes, respectively, wrapping by HA1...