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). Our work, together with others, could potentially provide a framework for antiviral drug and antibody development. He has Goku's softer jawline and Vegeta's sharper eyes. (A) Cryo-EM data processing flowchart. The “zero-out” density map was calculated by setting the density value within 3 Å around the atoms of the model to zero. The protease was removed by affinity purification using glutathione Sepharose 4B beads (GE Healthcare, 17-0756-01). Side view (left) and bottom-up view (right) are shown, respectively. The significant structure differences between the simulated coiled coil and the central helices also indicate differences in the coiled coil parameters of the Helix Cs and the Helix Ds. No, Is the Subject Area "Antibodies" applicable to this article? This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. (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. Upon virus entry, HA is triggered by low pH and undergoes large structural rearrangements from a prefusion state to a postfusion state. The supernatant containing the HA ectodomain was collected by centrifugation 48 hours posttransfection. Semitransparent surfaces displayed with the backbone ribbons in the density maps. https://doi.org/10.1371/journal.ppat.1009062.s015, https://doi.org/10.1371/journal.ppat.1009062.s016, https://doi.org/10.1371/journal.ppat.1009062.s017. The map is contoured at 8 σ. The surface grooves and the larger inner cavity in the stem region of the late intermediate conformation could provide new sites for designing universal vaccines and inhibitors to influenza viruses. 1021 GIFs. For the dataset of the HA-Fab complex at pH 7.8, 567,365 particles were selected after several rounds of 2D classifications. 12.8K views | Created Mar 03, 2019. gaming rocketleague rocket league. The two HA protomers in the trimer are colored dark grey and light pink color, respectively. The Helix Ds are more open and have a larger twist compared to the simulated coiled coil. Preparation of the antibody expression plasmids followed the same procedure as for HA expression. 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). Particles were automatically selected using Gautomatch [41]. The fusion peptides are colored green. The pH of the purified ecto HA-Fab complex sample was adjusted to 5.2 by the addition of 0.5 M sodium acetate at pH 4.6. We thank the Tsinghua University Branch of the China National Center for Protein Sciences (Beijing) for providing the facility support. The recombinant HA was eluted with the balancing buffer containing 5 mM d-desthiobiotin (Sigma, D1411). Through initial biochemistry and cryo-EM data analyses (S1 and S2 Figs), we found that F005-126, a neutralizing antibody targeting the HA head of H3N2 viruses, fulfills all our requirements. (A) The epitope of 31.a.83 is shown in a space-filling model. The cell membrane was collected by ultracentrifugation at 100,000×g for 1 hour at 4°C. The three HA1/HA2 heterodimers are colored hot pink, cornflower blue and orange, respectively. The 3D classifications and refinements were conducted with the C3 symmetry imposed. 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 structure elements are labeled and colored the same as in (A). 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. Upon virus entry, HA is triggered by low pH and undergoes large structural rearrangements from a prefusion state to a postfusion state. In summary, we reported two intermediate states of influenza HA at the low pH-induced transition, providing structural snapshots of HA in the highly dynamic process during virus-host cell membrane fusion. The best GIFs are on GIPHY. A ten question quiz to assign you a quirk, as in MHA. Sphericities indicate the degree of anisotropy present in the reconstructions. The fusion peptide wraps around the N-terminal fragment of HA1 (residues 9–19 of HA1, HA1-N) and forms a hydrogen bond with residue His17 of HA1-N. Hydrophilic pockets are formed between the Helix Ds (Fig 1D and 1E). (B) 31.a.83 binding to the low pH treated HA. Different conformations were observed. Further analysis showed that conformational changes occur mainly in the Helix Ds, as indicated by the calculated r.m.s.d values (0.4 Å between the 90 aligned Cα atom pairs of the Helix Cs vs 4.0 Å between the 60 aligned Cα atom pairs of the Helix Ds, Fig 3A). Visualization, Right, pH 5.2 conformation C. The directional FSC plots for the reconstructions are calculated on the 3DFSC server. The purified HA and Fab were mixed at a molar ratio of 1:3 (HA: Fab). #freddy The “zero out” residue density maps are colored yellow and contoured at 7 σ (D), 11 σ (E) and 7 σ (F), respectively. Search millions of user-generated GIFs Search millions of GIFs Search GIFs. These observations suggest that dropping the pH induced the release of the fusion peptides, which was also indicated by the aggregation of the particles (S2 and S3 Figs). For the dataset of the HA-Fab complex at pH 5.2, 579,459 particles were selected after several rounds of 2D classifications. It also creates a similar being that wears Metamoran attire like the Fusion Dance. In the present research, by using cryo-EM and 3D classification, we determined the structures of HA in low pH induced intermediate states. Low pH treatments were taken under 37°C. The extraction was then ultracentrifuged at 150,000×g for 30 minutes. The backbone of the fusion peptide is colored green, and the sidechains of the hydrophobic residues are colored gray. When he possesses Jaden, he dons his aforementioned outfit and Jaden's eyes becom… For one liter of cell culture, 2 mg of purified plasmid was preincubated with 8 mg of polyethylenimine (PEI) (Polysciences, 24765–2) for 20 minutes, and then the cells at a density of 2.5–3×106 cells/ml were transiently transfected with the plasmid-PEI mixture. The antigen-binding fragment (Fab) of F005-126 was prepared by protease cleavage of F005-126, and the HA-fab complex was assembled in vitro from purified Fab and HA. Upload Create. 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. Karoly's appearance is a mixture of Broly's Legendary Super Saiyan form and Goku's Super Saiyan form. The supernatant was discarded, and the cell pellet was resuspended in lysis buffer (20 mM HEPES pH 7.8, 150 mM NaCl) and sonicated. The conserved residues are boxed. The bound Fab F005-126 prevents the pre-postfusion transition of the HA and renders the HA trypsin resistant. Yes The stem specific antibody 31.a.83 in full length was used in the ELISA assay instead of its Fab part. of 0.5 Å between the 1470 aligned Cα atom pairs of the HAs. (D) Surface-rendered diagrams showing the changes in surface and inner cavity of the central helices upon pH change. https://doi.org/10.1371/journal.ppat.1009062.s007, https://doi.org/10.1371/journal.ppat.1009062.s008. (A) Size distribution of the HA-Fab complex at pH 7.8. https://doi.org/10.1371/journal.ppat.1009062.s014. Variations in local resolution were estimated by using RELION. Further cryo-EM analysis of the sample showed that the HA (strain A/Hong Kong/1/1968) with the bound Fab F005-126 was mostly intact at pH 5.2 (S2 Fig). The expression and purification of 31.a.83 was similar to that of F005-126. 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]. Search, discover and share your favorite Ha GIFs. The central helices of the pH 7.8 conformation (cornflower blue), the pH 5.2 conformation C (hot pink) and the headless HA (PDB accession number: 5CJQ), which could represent different stages in the pre-post transition, are compared. The surface is colored from white to green to purple according to the distances from the voxels to the three-fold axis. Similar tufts line the bottom of its face and two singular ones form its ears. 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. Subsequent 2D and 3D analyses were performed using RELION [42]. PLOS Pathogens publishes Open Access research and commentary that significantly advance the understanding of The shift of the C terminus is approximately 15 Å and the shift of the center of mass is approximately 7 Å. The HA head, after being stabilized by an antibody, remains unchanged compared to the neutral pH state. The rotation of the C terminus with the membrane-distal end of each central helix as the pivot point (residue Arg76) is approximately 9.5 degrees. (B) Structure comparisons between the Helix Ds in neutral (cornflower blue) and low pH (hot pink) conformations showing the changes in the residue side chains. Yes One of these classes showed similar overall features to those of the pH 7.8 conformation except for the fusion peptides. 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). Histogram indicates the portion of voxels with a particular resolution. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The N-terminal Helix Cs are tightly packed through extensive hydrophobic interactions (Fig 1D). Great for OCs. The supernatant was collected and applied to Strep-Tactin Superflow beads (IBA, 2-1206-010), which were precooled at 4°C and balanced with a buffer containing 20 mM HEPES at pH 7.8, 150 mM NaCl and 0.003% LMNG. The completely conserved residues are shown in white on a red background. (C) A schematic diagram illustrating the low pH-induced structural transition of HA. It has a short, dark brown snout and a small, red nose. Atomic models of the HA ectodomain and the bound Fabs were built (Fig 1B and 1C). The N-terminal hydrophobic residues of the fusion peptide are shown as orange spheres. Residues not conserved are colored white. As the most important antigen of influenza virus, hemagglutinin (HA) is a primary target of drugs and vaccines. In addition, wrapping by the HA1 head should further stabilize the membrane-distal Helix Cs. The cleaved free Fc and the uncleaved F005-126 were removed using a protein A Sepharose column. The coiled-coil parameters of the Helix Cs and Helix Ds in different conformations were calculated and compared. 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]. Visualization, Residue side chains are shown in balls and sticks with oxygen atoms colored red, nitrogen atoms colored blue and sulfur atoms colored yellow. Cavities colored blue are large enough to adapt more than two water molecules [53]. The central helices are colored cornflower blue. The best GIFs are on GIPHY. 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). 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). 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). Are you ready to find out your quirk? Manhattan District The Trinity test of the Manhattan Project on 16 July 1945 was the first detonation of a nuclear weapon. To signify his position as the Supreme King, he also wears a black helmet that functions as a crown. 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). (A) Structure comparisons of the Helix As in neutral and low pH conformations. (D to F) Surface-shadowed and ribbon diagrams showing the residue densities of the “zero out” maps for the three low pH conformations. 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 sample was incubated at room temperature for 30 minutes and was then loaded directly onto grids for cryo-EM sample preparation. See Materials and methods for details. Middle, pH 5.2 conformation B. Yes No, Is the Subject Area "Crystal structure" applicable to this article? 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). The synthesized HA gene was cloned into the pCMV vector, which introduced a strep tag at the C-terminus of the recombinant HA. 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 density map of the HA-Fab complex at pH 7.8 was used as the initial model. The grids were blotted and then plunged into liquid ethane by using a Vitrobot Mark IV (Thermo Fisher). ieGod @iegod. It’s likely that our strategy of using a HA head specific antibody helps to stabilize this intermediate state which is then captured by cryo-EM. values between the 813 aligned Cα atom pairs of HA heads (residues 43–313 of HA1) of pH 5.2 conformation A, pH 5.2 conformation B and pH 5.2 conformation C with the conformation at pH 7.8 are 0.25 Å, 0.53 Å and 0.36 Å, respectively. After concentration and buffer exchange, the supernatant was loaded onto Strep-Tactin Superflow beads. (D) Ribbon and surface diagrams showing the central helices. The profiles of the disordered transmembrane domain and the bound detergent micelle (map contoured at 3 σ) are indicated by brown lines. 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. Our research revealed structural details of HA intermediates upon low pH treatment, deepening our understanding of virus entry, potentially providing new hints for universal flu therapy and vaccines. The three Helix Ds are tethered together at the membrane-proximal ends, which bend and rotate anticlockwise by 3.5 degrees compared to a modeled coiled coil generated from the parameters of the Helix Cs (S6 Fig). 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. https://doi.org/10.1371/journal.ppat.1009062.s002. The neutral pH sample is in a buffer containing 150 mM NaCl, 20 mM HEPES at pH 7.8 and 0.003% LMNG and the complex is at a concentration of 0.07 mg/ml. 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. 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. https://doi.org/10.1371/journal.ppat.1009062.s010. The beads were washed with balancing buffer. The eluted sample was concentrated and treated with trypsin (Sigma, T1426) for 12 hours in a buffer containing 50 mM Tris-HCl at pH 8.0, 20 mM CaCl2 and 0.003% LMNG. Scattering data were analyzed with the software DYNAMICS 7.1.7. Residues in blue belong to Helix A. https://doi.org/10.1371/journal.ppat.1009062.s012. (D) 31.a.83 binding to the low pH treated HA-Fab F005-126 complex. The densities around the stem region of the HA-Fab-pH 5.2 conformation C are highly disordered, and atomic models were built only for the head and the central helix (Figs 2C, S7 and S10D). All the measurements were repeated three times. https://doi.org/10.1371/journal.ppat.1009062, Editor: Félix A. Rey, Institut Pasteur, FRANCE, Received: July 26, 2020; Accepted: October 9, 2020; Published: November 30, 2020. 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 zoomed-in views show the densities around the fusion peptides. Side view (left) and bottom view (right) are shown. 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. After concentration and buffer exchange, the supernatant was loaded onto a protein A Sepharose column (GE Healthcare, 17-0780-01). Splaying of the helices were also observed in two trimer tag stabilized HA2 structures [27,28], in which the Helix Ds has a splaying angle of approximately 30 degrees [27] (S11 Fig) and may present an intermediate to the postfusion state or an irrelevant state caused by the trimer tag. The purified and trypsin cleaved HA was coated directly onto a 96-well ELISA plate overnight at 4°C. https://doi.org/10.1371/journal.ppat.1009062.s006. 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. (B) Local resolution map. 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. Zoom-in views of the fitted region are shown at the right. The amount of HA loaded in each lane was adjusted to be the same. Search, discover and share your favorite Gem Fusion GIFs. e1009062. Fusion HA! 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). Find GIFs with the latest and newest hashtags! 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. Mar 6, 2016 - Discover the magic of the internet at Imgur, a community powered entertainment destination. The fusion peptide (residues 1–20 of the HA2) was excluded for all the calculations. 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. The steric clashes between the models were calculated with Chimera (Fig 4). Vegito is a mixture of both Goku and Vegeta's characteristics, with a few deviations. Our study showed that after being stabilized by Fab F005-126, the interface between the HA1 globular head and Fab F005-126 remained intact even after low-pH treatment for 30 minutes (S5 Fig). Residues not conserved are colored white. The membrane pellet was resuspended with 1% (w/v) lauryl maltose neopentyl glycol (LMNG) (Anatrace, NG310) in lysis buffer and incubated at 4°C for 2 hours. Search, discover and share your favorite Fusion Ha GIFs. The second form in that paper is quite consistent with our conformation C, as they both feature with unwound central helices. 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. Investigation, Here, we report two distinct low pH intermediate conformations of the influenza virus HA using cryo-electron microscopy (cryo-EM). Similarly, the extended Helix D would have steric clashes with the outer Helix A, Hairpin 2 and HA1-N (Fig 4), suggesting that Helix A and the β sheet constituted by Hairpins 1 and 2 and HA1-N must undergo conformational changes to adapt to the extended Helix D. The densities around the Helix A indicate that its C-terminal end should have a clockwise shift (Figs 5A and S14). Particles from classes showing obviously different conformations in the stem region were combined and subjected to another round of 3D classification, yielding six classes. Hairpin 2 (126–141), purple. TMB (3,3′,5,5′-tetramethylbenzidine) substrate (CWBIO, CW0050S) was used for the horseradish peroxidase-based detection and the reaction was stopped by adding H2SO4 with the concentration of 0.5 M. Signal of the horseradish peroxidase product was detected by measuring their absorbance at 450 nm. https://doi.org/10.1371/journal.ppat.1009062.s004. Right: Zoomed-in views showing the Helix As in neutral (red) and low pH (yellow) conditions. (D) Directional FSC plot for the reconstruction calculated on the 3DFSC server. (A) Changes in the pore radius along the Z axis. With Tenor, maker of GIF Keyboard, add popular Fusion animated GIFs to your conversations. Validation, Both the increase in the superhelical radius and the decrease in the superhelical frequency of the Helix Ds indicate the unwinding of the coiled coil. Images were recorded at a defocus range of -1 μm to -3.5 μm with a pixel size of 0.665 Å. A year on, we finally got the courage to try again...with horrible results. https://doi.org/10.1371/journal.ppat.1009062.s009. (C) Ribbon diagrams showing the structure elements of a HA1/HA2 heterodimer. 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. Data collection and image processing statistics are listed in S1 Table. The transmembrane domain of the HA is flexible and was excluded from the final structure model (Figs 1B and S4). The surface is colored according to the surface electrostatic potential with positive charges colored blue and negative charges colored red. Thus, we fitted the stem region of the pH 7.8 structure into the density map of the pH 5.2 conformation C as a rigid body (Fig 4). The conformation B has the released fusion peptides while the other domains remain in similar conformation with the neutral pH structure. Trypsin was then added to the samples at a ratio of 1:20 (trypsin:HA, w/w). The interactions between the helices observed in the neutral conformation are disrupted (Fig 3B). Each image was dose-fractionated into 32 movie frames at a dose rate of 8.2 counts per physical pixel per second, with a total exposure time of 8 s and a frame exposure time of 0.25 s, resulting in a total dose of ~50 electrons per Å2. The bound Fabs are colored tan. Then, two parallel rounds of 3D classification were conducted, yielding six classes and five classes, respectively. https://doi.org/10.1371/journal.ppat.1009062.g001. The buffer used was 20 mM HEPES at pH 7.8 with 150 mM NaCl. Yes (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). Left, pH 5.2 conformation A. 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. Eluted sample was adjusted to pH 7.8 and 150 mM NaCl S1 and S2 Tables decrease of the Middleware. 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