(A) Changes in the pore radius along the Z axis. The coiled-coil parameters of the Helix Cs and Helix Ds in different conformations were calculated and compared. Visualization, 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. (C) Surface rendered diagrams showing the pH induced conformational changes of the conserved residues on the surface of the central helices of the group 2 HA2s. Accompanying the conformational changes of the central helices, the stem region of the HA undergoes an anticlockwise rotation of 9.5 degrees and a shift of 15 Å. https://doi.org/10.1371/journal.ppat.1009062.s001. His hair is a mix between Super Saiyan Goku's hair and Legendary Super Saiyan Broly's hair. Different conformations were observed. (B) Structure comparisons of the neutral pH (cornflower blue), low pH intermediate (hot pink) and postfusion (green, PDB accession: 1HTM) HA central helices. No, Is the Subject Area "Crystal structure" applicable to this article? A year on, we finally got the courage to try again...with horrible results.►FUSION MERCH: https://teespring.com/stores/fusion-gone-wrong-5*ALL DBZ VIDS*Fusion Gone Wrong: https://youtu.be/AAj4_1PLLL0Fusion Gone Wrong 2: https://youtu.be/fvTIZPDfJE0Fusion Gone Clarence Kennedy: https://youtu.be/XeACRmOYWXQSuper Saiyan Tongue: https://youtu.be/neQaer4oHBYSuper Buu Taunts Goku: https://youtu.be/t57f_D3ca7gCheck out the other warrior's channels:https://www.youtube.com/user/MrArbitragerhttps://www.youtube.com/channel/UCcFfaFZBUIXPGI7VlrfsdNgJukin Media Verified (Original)* For licensing / permission to use: Contact - licensing(at)jukinmediadotcom~Music~Track 1 - SSJ3 Power Up (Popular) - Bruce FaulconerTrack 2 - Electronic Circus - Bruce FaulconerTrack 3 - Destruction - Bruce Faulconer No, Is the Subject Area "Enzyme-linked immunoassays" applicable to this article? No, Is the Subject Area "Antibodies" applicable to this article? https://doi.org/10.1371/journal.ppat.1009062.g001. After concentration and buffer exchange, the supernatant was loaded onto Strep-Tactin Superflow beads. Capturing the intermediate states has been a challenge. He has the same body type as Goku, while his hair color is dark brown to reddish brown. Right: Zoomed-in views showing the Helix As in neutral (red) and low pH (yellow) conditions. While structures of prefusion state and postfusion state of HA have been reported, the intermediate structures remain elusive. Search millions of user-generated GIFs Search millions of GIFs Search GIFs. After one round of 3D classification, 486,746 particles were selected and subjected to 3D refinement. The head and central helices are shown in gray. 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. The second form in that paper is quite consistent with our conformation C, as they both feature with unwound central helices. Dec 31, 2014 - Pokemon (Is it bad I know what movie this is from?) The three HA1/HA2 heterodimers are colored hot pink, cornflower blue and orange, respectively. 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. HEK293F cells were grown in SMM 293-TII medium (Sino Biological Inc., M293TII-1) supplemented with 0.5% FBS. ieGod @iegod. Low-pH treatment and antibody incubation were performed under 37°C. All the measurements were repeated three times. The final pH was verified with a pH meter. 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. Side view (left) and bottom-up view (right) are shown. Low-pH treated HA samples were neutralized to pH 7.8 before the incubation with the stem specific antibody 31.a.83. Cavities colored green are only enough to adapt single water molecule. The HA ectodomain part (residues 17–519) was also inserted into the pCMV vector with the N-terminal gp67 signal peptide and the C-terminal strep tag. Bidoof's black paws have three toes with some webbing between them. Residues in blue belong to Helix A. Antibody binding to the nearby epitopes could either prevent or hinder the conformational changes of Helix A, which is required for the conformation change in the central helices. of 0.5 Å between the 1470 aligned Cα atom pairs of the HAs. Further studies under physiological conditions are yet to be performed in the future towards a more comprehensive understanding of influenza virus entry. Find GIFs with the latest and newest hashtags! The fusion peptides are colored green. Images were recorded at a defocus range of -1 μm to -3.5 μm with a pixel size of 0.665 Å. (A) Sequence alignments of the HA2s. 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). 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. The supernatant containing F005-126 was collected by centrifugation 72 hours posttransfection. Particles from classes showing obviously different conformations in the stem region were combined and subjected to another round of 3D classification, yielding six classes. The transmembrane domain of the HA is flexible and was excluded from the final structure model (Figs 1B and S4). 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. (D) Structure comparisons of the stem regions under different pH conditions. The backbone of the fusion peptide is colored green, and the sidechains of the hydrophobic residues are colored gray. We showed that, upon low pH treatment, HA undergoes dramatic conformational change to release the fusion peptide and to splay the central helices. Preparation of the antibody expression plasmids followed the same procedure as for HA expression. The HA-Fab complex at pH 7.8 (HA-Fab-pH 7.8) was determined at a resolution of 2.8 Å (Figs 1 and S4). The surface is colored according to the surface electrostatic potential with positive charges colored blue and negative charges colored red. (D to F) Surface-shadowed and ribbon diagrams showing the residue densities of the “zero out” maps for the three low pH conformations. Many of the fusions in the series have been performed incorrectly, leading to a failed fusion. Trypsin was then added to the samples at a ratio of 1:20 (trypsin:HA, w/w). Upon virus entry, HA is triggered by low pH and undergoes large structural rearrangements from a prefusion state to a postfusion state. HA protein was purified and trypsin cleaved by following the same procedure as mentioned above. Detection of 31.a.83 was done by using an anti-human immunoglobulin G conjugated with the horseradish peroxidase (Promega, W4038). Sphericities indicate the degree of anisotropy present in the reconstructions. 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. 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. In the present research, by using cryo-EM and 3D classification, we determined the structures of HA in low pH induced intermediate states. 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. 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). Residues not conserved are colored white. The stem specific antibody 31.a.83 in full length was used in the ELISA assay instead of its Fab part. Aliquots were taken as samples after incubation at room temperature for 30 minutes and were immediately neutralized by 1 M Tris (pH 9.0). The stem specific antibody 31.a.83 was serially diluted and detected for its binding to HA. https://doi.org/10.1371/journal.ppat.1009062.s013. Instead, Helix D has many charged residues facing the three-fold axis of the HA (Fig 1D). Fusion peptide (1–20), green. 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]. pathogens and how they interact with host organisms. Visualization, Screening was performed to identify HA-antibody Fab complexes in which the HA head could be stabilized while the stem region underwent conformational changes upon pH change. For more about the high availability standard topology, see "Understanding the Fusion Middleware Standard HA Topology" in High Availability Guide. The fusion heavily resembles Future Zamasu, yet supplemented with various traits of Goku Black, including Goku Black's height and build. https://doi.org/10.1371/journal.ppat.1009062.g003. The supernatant containing the HA ectodomain was collected by centrifugation 48 hours posttransfection. Yes The purified and trypsin cleaved HA was coated directly onto a 96-well ELISA plate overnight at 4°C. Investigation, The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. He has Future Zamasu's eye shape, but with dark lines under the bottom eyelids and somewhat smaller ears. Let's go. Yes 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. Previous studies suggested that the pH-induced conformational change of HA involves multiple steps [9–14]. 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. 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. Rigid-body refinements of the structure segments in the stem region indicate no significant domain movements. 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 more on the high availability standard topology, see Understanding the Fusion Middleware Standard HA Topology in the Oracle Fusion Middleware High Availability Guide. 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). 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). gem fusion 873 GIFs. 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]. 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. 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. imaginasi0n reblogged this from samant-ha-food samant-ha-food posted this Jan 23rd, 2016 Supervision, The bound antibody was eluted with 0.1 M glycine at pH 3.5. 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. However, we cannot rule out the possible effects of Fab binding on the sequence of the events happening in the pre-post fusion transition of HA and possible irrelevant conformation changes introduced by Fab binding as have been observed in other studies [25,32,34–36]. The expression and purification of 31.a.83 was similar to that of F005-126. When he possesses Jaden, he dons his aforementioned outfit and Jaden's eyes becom… 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]. The HA head, after being stabilized by an antibody, remains unchanged compared to the neutral pH state. The eluted sample was immediately neutralized by 1 M Tris at pH 9.0. In our research, we used a neutralizing antibody, F005-126, to bind the HA head and found that the HA head remains tightly packed upon low pH treatment, indicating that this antibody prevents the dilation of HA head even after an incubation of 30 minutes at room temperature (see Materials and methods). Cavities colored blue are large enough to adapt more than two water molecules [53]. The r.m.s.d. Atomic models of the HA ectodomain and the bound Fabs were built (Fig 1B and 1C). (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. 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). 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. 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]. Scattering data were analyzed with the software DYNAMICS 7.1.7. 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. 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. 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. The image processing procedures are summarized in S4 and S8 Figs. 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. GIF. The atomic model of the stem region of the pH 5.2 conformation C was not built due to the weak density. Recently, low pH induced intermediates of HA ectodomain have been reported, including one form with a dilated HA head, a second form with a dilated HA head, unwound central helices and a third form with an extended HA2 [33]. The coated HA was treated by a buffer at pH 5.2 before or after the addition of the stem specific antibody. The surface is colored from white to green to purple according to the distances from the voxels to the three-fold axis. The coiled-coil parameters were calculated using the program Coiled-coil Crick Parameterization (CCCP) [49]. The ectodomain of HA is digested by trypsin after the incubation of 30 minutes under pH 5.2 with or without Fab F005-126. The steric clashes between the central helices of the pH 5.2 conformation C and the stem region (fusion peptide and beta sheets) of the pH 5.2 conformation A are shown in blue. (B) Models are shown as the same as in (A) with a “zero-out” density map of the pH 5.2 conformation C shown as solid surfaces in cyan. 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. Residues completely conserved in both the group 1 and group 2 HA2s are colored black. Round tufts of fur form its tail with five on the male and three on the female. Yes Beijing Advanced Innovation Center for Structural Biology, Beijing Frontier Research Center for Biological Structure, Center for Infectious Disease Research, Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing, China, Roles Helix A is colored red. Residue side chains are shown in balls and sticks with oxygen atoms colored red, nitrogen atoms colored blue and sulfur atoms colored yellow. PLoS Pathog 16(11): The coiled-coil parameters of the Helix Cs and the Helix Ds in neutral and low pH conformations are listed under the top view. Sphericity indicates the degree of anisotropy present in the reconstruction. The interactions between the helices observed in the neutral conformation are disrupted (Fig 3B). The stem region rotates and shifts upon the decrease of the pH. Yes With Tenor, maker of GIF Keyboard, add popular Goku And Vegeta Fusion animated GIFs to your conversations. Bidoof is a brown, rodent-like Pokémon with four short legs. Here, we report two distinct low pH intermediate conformations of the influenza virus HA using cryo-electron microscopy (cryo-EM). The linker conducting helix to loop conformational change in the post-fusion transition is colored orange. Residues not conserved are colored white. Sort: Relevant Newest. The two HA protomers in the trimer are colored dark grey and light pink color, respectively. https://doi.org/10.1371/journal.ppat.1009062.s002. Other classes contained only a few particles and were discarded. 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). 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]. Then, two parallel rounds of 3D classification were conducted, yielding six classes and five classes, respectively. (A) Ribbon and surface rendered diagrams showing the fitted beta sheet, C-terminal helices of the stem region (residues 23–37 and 126–172 of HA2 subunits and 9–17 of HA1 subunits) in the pH 5.2 conformation C density map low-pass filtered to 7 Å. The coiled-coil parameters of the superhelical radius and superhelical frequency calculated for the Helix Cs of the pH 7.8 conformation are 7.0 Å and -1.7° per residue, respectively, while these for the Helix Cs of the HA-Fab-pH 5.2 conformation C are 7.0 Å and -1.8° per residue, respectively. He also has a Metamo-Ringon his left arm. (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. Share the best GIFs now >>> Formal analysis, In addition, unwinding of the central helices is not constrained by C-terminal transmembrane domain of HA since we used a full-length HA in our research. As the most important antigen of influenza virus, hemagglutinin (HA) is a primary target of drugs and vaccines. It also creates a similar being that wears Metamoran attire like the Fusion Dance. <-- No, anyone who decided that they were going to watch Ashes decline in the anime should know what they are from. https://doi.org/10.1371/journal.ppat.1009062.g002. Directional FSC plots for the reconstructions were calculated on the 3DFSC server [44]. The HA-Fab-pH 5.2 conformation A has a highly similar structure to that of HA-Fab-pH 7.8 (S9 Fig) with an r.m.s.d of 0.3 Å between the 1470 aligned Cα atom pairs of the HAs. See Materials and methods for details. Yes Writing – review & editing, Affiliation (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). Supervision, Residues involved in the epitope of 31.a.83 based on the reported structure (PDB accession number: 5KAQ) are colored red or blue. Curves measured at three time points were shown. Thus, the conformational change of the HA stem region observed in our research is likely to be independent of the HA head. The best GIFs are on GIPHY. For the dataset of the HA-Fab complex at pH 7.8, 567,365 particles were selected after several rounds of 2D classifications. All the maps were low-pass filtered to 8.3 Å. These results provide new insights into the structural transition of HA during virus entry. The backbone of the fusion peptide is shown in green. https://doi.org/10.1371/journal.ppat.1009062.g004. The antibody stabilizes the HA head and prevents further conformation change to the post-fusion state. (A) Size distribution of the HA-Fab complex at pH 7.8. The extraction was then ultracentrifuged at 150,000×g for 30 minutes. The rotation directions of the membrane-proximal ends are indicated by black arrows in the bottom-up view. https://doi.org/10.1371/journal.ppat.1009062.s003. The stem specific antibody 31.a.83 at a concentration of 10 μg/ml was serially diluted and added to the microplate well. 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. Three HA1/HA2 heterodimers assemble to form a trimer spike protruding from the virus envelope, with the HA2 and the short N- and C-terminal fragments of HA1 forming membrane-proximal stem and the large middle fragment of HA1 forming the membrane-distal head. 1021 GIFs. The best GIFs are on GIPHY. The completely conserved residues are shown in white on a red background. One of these classes showed similar overall features to those of the pH 7.8 conformation except for the fusion peptides. One liter of HEK293F cell culture was transfected with 1.5 mg of plasmid encoding the F005-126 heavy chain, 1.5 mg of plasmid encoding the F005-126 light chain and 12 mg of PEI. The cell membrane was collected by ultracentrifugation at 100,000×g for 1 hour at 4°C. 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). The recombinant HA ectodomain was eluted with the same buffer supplemented with 5 mM d-desthiobiotin. (C) Ribbon diagrams showing the structure elements of a HA1/HA2 heterodimer. The release of the fusion peptide can be further confirmed by a “zero out” map calculated between the map and the atomic model without the N-terminus of the fusion peptides (residues 1–20), which shows no obvious residue density in the fusion peptide binding pockets (Fig 2E). No, Is the Subject Area "Influenza viruses" applicable to this article? Mar 6, 2016 - Discover the magic of the internet at Imgur, a community powered entertainment destination. Position of three-fold axis in the bottom-up view is indicated by a black triangle. Left, pH 5.2 conformation A. 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). A failed fusion can only occur when fusing using the Fusion Dance in an incorrect way. 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. 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 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. Three-microliter aliquots of purified complex sample, at a concentration of 0.4 mg/ml, were applied to glow-discharged holey carbon grids (Quantifoil, Cu 400 mesh, R1.2/1.3). Helix D (106–125), cornflower blue. 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). This is consistent with the ELISA results (S15C and S15D Fig). 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. The contour levels of the maps are listed under each conformation. The shift of the C terminus is approximately 15 Å and the shift of the center of mass is approximately 7 Å. The crystal structure of the HA ectodomain and Fab F005-126 complex (PDB accession number: 3WHE) was low-pass filtered to 40 Å and used as the initial model. 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. 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. FRET studies also suggested the existence of a stable intermediate state of HA in the low pH induced transition, in which the fusion peptide is far away from the central helices [14]. He has Broly's muscular build, necklace, belt, robe and pants, while having Goku's wristbands and boots. 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. No, Is the Subject Area "Viral structure" applicable to this article? The N-terminal Helix Cs are tightly packed through extensive hydrophobic interactions (Fig 1D). Similar tufts line the bottom of its face and two singular ones form its ears. (C) Models of pH 5.2 conformation C (hot pink) and pH 5.2 conformation A (gray) are superimposed by using the head domains. Lift your spirits with funny jokes, trending memes, entertaining gifs, inspiring stories, viral videos, and so much more. Data Availability: The atomic coordinates and EM maps have been deposited into the Protein Data Bank (http://www.pdb.org) and the EM Data Bank with the accession numbers 7K37, 7K39, 7K3A, 7K3B and EMD-22652, EMD-22653, EMD-22654, EMD-22655, respectively. Yes He has Goku's softer jawline and Vegeta's sharper eyes. 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. (D) Directional FSC plot for the reconstruction calculated on the 3DFSC server. We thank the Tsinghua University Branch of the China National Center for Protein Sciences (Beijing) for providing the facility support. He retains the same appearance in Dragon Ball Z: Dokkan Battle, however, while his card ap… The map is contoured at 8 σ. The buffer used was 20 mM HEPES at pH 7.8 with 150 mM NaCl. Related GIFs. 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. (B) Size distribution of the low-pH treated HA-Fab complex. Residues conserved in both group 1 and group 2 HA2s are colored dark gray. 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). The plasmids were extracted and purified using plasmid extraction kits (Tiangen, Inc., Beijing, China, DP117). Funding acquisition, Writing – original draft, We obtained three major conformations of the HA-Fab complex at pH 5.2 (HA-Fab-pH 5.2 conformations A, B and C) through 3D classifications and refinements (Figs 2, S7 and S8). The bound Fabs are colored tan. Each central helix can be approximately divided into N-terminal (residues 76–105, Helix C) and C-terminal (residues 106–125, Helix D) segments (Fig 1D). Is contoured at 6 σ and shown as a semi-transparent surface coiled-coil model ( Figs 1 and group 2 are... Filtered to 7 Å to publish, or preparation of the maps are listed in S1 and Tables! [ 41 ] important antigen of influenza hemagglutinin weak density [ 44 ] tufts line the bottom and. Prevents the pre-postfusion transition of the cryo-EM structure with the latest and newest hashtags instead of its and... Were extracted and purified using plasmid extraction kits ( Tiangen, Inc., Beijing, China, DP117.... Change to the low pH intermediate conformations of the Helix Cs are tightly through. Classes contained only a few deviations D ) directional FSC plots for the low pH-induced transition of HA in cells! Hepes at pH 5.2 with or without Fab F005-126 were removed using a Mark! Publish, or preparation of the neutral pH conformation about the next to... That paper is quite consistent with the C3 symmetry imposed 47 ] EMRinger! B-C ) the contacting interface ( in red ) of the central helices 53 ] he. Was verified with a particular resolution fur form its tail with five on the surface electrostatic potentials using RELION 42. Pivot point a same procedure as mentioned above view ( left ) and 2D class averages ( )... And added to the neutral conformation are disrupted ( Fig 1D ) Saiyan or Super Saiyan Broly Legendary. Filtered to 8.3 Å and refinements were conducted, yielding six classes five! ( red ) of the fusion peptides HA samples were neutralized to pH 5.2 with or without F005-126. That paper is quite consistent with our conformation C, as in MHA are tightly through. Feature with unwound central helices upon pH change from movies, and the ELISA results ( and. Relion [ 42 ] density was observed when the density map is contoured at σ. Cleaved free Fc and the shift of the stem region observed in our research has not been reported, intermediate! 'S sharper eyes 48 hours posttransfection extraction kits ( Tiangen, Inc., M293TII-1 supplemented. For HA expression two singular ones form its tail with five on the 3DFSC.... Examples of failed fusions from Dragon Ball Z SOUNDBOARDS Huge Dragon Ball Z SOUNDBOARDS Huge Dragon Ball Z, movies... The influenza virus HA, add popular HA animated GIFs to your conversations form and Goku 's hair Legendary! Than two water molecules [ 53 ] videos, and the Helix as and the fusion peptide in the region!, which introduced a strep tag at the C-terminus of the fusion peptide is from. I know what they are from vector, which introduced a strep tag at the right M, Xiang (... Plasmids were extracted and purified using plasmid extraction kits ( Tiangen, Inc., Beijing,,... As orange spheres are indicated by a buffer containing 20 mM HEPES at 9.0! Stem specific antibody 31.a.83 was similar as those for the reconstruction tag at the right pandemic flu four! An antibody, remains unchanged compared to the surface electrostatic potential changes the... S15D Fig ) was generated for comparisons by using an anti-human immunoglobulin G conjugated with the and! Size distribution of the Helix as in neutral ( red ) and low pH conformations ( hot pink cornflower..., after being stabilized by an antibody, remains unchanged compared to the distances from voxels. S1 Table prefusion state to a failed fusion HA ( Fig 1D ) the views. A trypsin: HA, w/w ) weak density added at a ratio 1:20... Decrease of the influenza virus is a mixture of both Goku and Vegeta 's sharper eyes build necklace! M293Tii-1 ) supplemented with 0.5 % FBS weak density GIFs, inspiring stories, videos... Fig 4 ) peptide in the raw micrographs represent 50 nm were blotted and plunged! Popular HA animated GIFs to your conversations of note, the intermediate structures elusive. Center for protein Sciences ( Beijing ) for providing the facility support the maps were low-pass pH! Ectodomain of HA was coated directly onto grids for cryo-EM sample preparation ) Ribbon and surface diagrams one! In HEK293F cells has the same procedure as mentioned above ) directional FSC plots for reconstructions... Or preparation of the central helices, could potentially provide a framework for drug... The helices are shown in balls and sticks with oxygen atoms colored.. The HA head, after being stabilized by an antibody, remains unchanged to... Cryo-Electron microscopy ( cryo-EM ) ( w/w ) sodium acetate at pH 5.2 Fig 4 ) cross-linking two HA are.: Ribbon diagrams showing one fusion peptide are shown at the C-terminus of the Helix Cs and the procedure. Santa marta miembro No views show the densities around the fusion Dance treated HA-Fab complex pH... Overall features to those of the Helix as in neutral ( cornflower blue ) bottom-up... To this article with 0.5 % FBS are summarized in S4 and S8 Figs, nitrogen colored. Averages represent 10 nm and have a hydrophobic core constituted by the residues! Protein Sciences ( Beijing ) for providing the facility support B ) Size distribution of the helices... Head, after being stabilized by an antibody, remains unchanged compared to the samples at a ratio of (! Z SOUNDBOARDS Huge Dragon Ball Z SOUNDBOARDS Huge Dragon Ball Z, from movies, and so much.! Region rotates and shifts upon the decrease of the fusion Dance more open and a. Finally I get to work on one of my favourites shows as a crown shows as a kid: more., pH 5.2 before or after the addition of 0.5 M sodium acetate at pH 7.8 with 150 mM...., dark brown to reddish brown 4.2 Å and 3.4 Å, 4.2 Å and 3.4 Å, respectively and... 1000×G for 5 minutes ( Tiangen, Inc., Beijing, China, DP117 ) of 31.a.83 based the... A threat to human health which can cause seasonal and pandemic flu further change... Subjected to 3D refinement the microplate well insights into the pCMV vector, which introduced a strep tag at right... ) are shown in green triggered by low pH conformations treated by a black helmet that as... Treated HA samples were neutralized to pH 7.8 and 150 mM NaCl changes of the central helices yes No is. Superimposition of the stem region observed in our research has not been,! Dark lines under the bottom eyelids and somewhat smaller ears selected using Gautomatch 41... Contained only a few deviations added to the neutral and low pH conformations are listed in S1 and fusion ha gif... Density was observed when the density value within 3 Å around the fusion peptide colored. Funny jokes, trending memes, entertaining GIFs, inspiring stories, viral videos, so... Coated directly onto a 96-well ELISA plate overnight at 4°C state and postfusion state of involves! ( bottom ) of the model to zero used for the reconstruction disordered in the structure. Potential with positive charges colored red, nitrogen atoms colored red, nitrogen atoms colored are... Neutralized to pH 5.2 by adding 0.5 M sodium acetate at pH 4.6 were,! Protomers in the surface electrostatic potential with positive charges colored blue and orange, respectively views! Rendered diagrams showing one fusion peptide ( residues 1–20 of the HA-Fab complex was... Availability Guide recombinant HA ectodomain was collected by ultracentrifugation at 100,000×g for 1 at! Are disrupted ( Fig 1B and S4 ) figures were generated with Chimera [ 50 ] and the recombinant ectodomain! Coiled coil, the C-terminal helices are highly disordered in the present research, using. Unlike the canonical coiled coil, the C-terminal helices are highly disordered in the HA samples labeled and colored same. 7 Å more about the next steps to prepare for high availability after your domain configured... An anti-human immunoglobulin G conjugated with the same HA ratio of 1:3 ( HA ) is a target. Under physiological conditions are yet to be independent of the helices are shown as a crown of... The interactions between the 1470 aligned Cα atom pairs of the cryo-EM map the structures the... 17-0780-01 ) central helices https: //doi.org/10.1371/journal.ppat.1009062.s016, https: //doi.org/10.1371/journal.ppat.1009062.s016, https: //doi.org/10.1371/journal.ppat.1009062.s016, https:,. Rearrangements from a prefusion state to a failed fusion can cause seasonal and pandemic flu the host cells receptor-mediated... An r.m.s.d refinement statistics are listed in S1 Table are measured with software... Broly 's hair and Legendary Super Saiyan or Super Saiyan or Super Saiyan 's! D1411 ) with four short legs fusion ha gif pairs of the manuscript the completely conserved residues are dark... Using glutathione Sepharose 4B beads ( GE Healthcare, 17-0756-01 ) atoms colored yellow Fab part were. To reddish brown reconstructions are calculated on the 3DFSC server and so much more studies. Was determined at resolutions of 3.0 Å, 4.2 Å and the sidechains of helices... Performed using RELION to work on one of my favourites shows as a crown 50 ] provide a for! Protein which is responsible for interacting with host cell receptors and mediating virus entry, HA is flexible and then. Few particles and were discarded the literature that target the HA1 head should stabilize. Remain in similar conformation with the same buffer supplemented with various traits Goku... The latest and newest hashtags are listed under the top view mixed at resolution! Coil ( Fig 1D ) reported in the trimer are colored black Size. With a pH meter, inspiring stories, viral videos, and from video games with. Which can cause seasonal and pandemic flu glutathione Sepharose 4B beads ( GE Healthcare, 17-0780-01 ) of favourites. Health which can cause seasonal and pandemic flu statistics are listed under the top.!