fusion ha gif
Writing – review & editing, Affiliation Upon virus entry, HA is triggered by low pH and undergoes large structural rearrangements from a prefusion state to a postfusion state. Right, pH 5.2 conformation C. The directional FSC plots for the reconstructions are calculated on the 3DFSC server. These results provide new insights into the structural transition of HA during virus entry. However, a bulky density was observed when the density map was low-pass filtered to 7 Å. 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. Left, pH 5.2 conformation A. Model building and structure refinement statistics are listed in S1 and S2 Tables. Dec 31, 2014 - Pokemon (Is it bad I know what movie this is from?) For each microplate well, 100 ng of HA was added. 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. In addition, the densities of β Hairpins 1 and 2, the C-terminus of the HA2 stem and HA1-N become smeared in the HA-Fab-pH 5.2 conformation B (S10 Fig), suggesting disorder in these regions. The structures of the three major conformations were determined at resolutions of 3.0 Å, 4.2 Å and 3.4 Å, respectively. The surface is colored from white to green to purple according to the distances from the voxels to the three-fold axis. 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. Copyright: © 2020 Gao et al. 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) . The backbone of the fusion peptide is colored green, and the sidechains of the hydrophobic residues are colored gray. All the maps were low-pass filtered to 8.3 Å. Helix A is colored red. To signify his position as the Supreme King, he also wears a black helmet that functions as a crown. The zoomed-in views show the densities around the fusion peptides. Helix A (39–56), blue. It also creates a similar being that wears Metamoran attire like the Fusion Dance. 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 . 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. 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 stem region rotates and shifts upon the decrease of the pH. 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. As the most important antigen of influenza virus, hemagglutinin (HA) is a primary target of drugs and vaccines. 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. Supervision, 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. The ectodomain of HA is digested by trypsin after the incubation of 30 minutes under pH 5.2 with or without Fab F005-126. 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. As indicated by our ELISA assays with the antibody 31.a.83 that mainly targets Helix A , the binding of 31.a.83 to HA was significantly reduced over the time course of low pH treatment (S15 Fig). gem fusion 873 GIFs. The sample was incubated at room temperature for 30 minutes and was then loaded directly onto grids for cryo-EM sample preparation. Finally I get to work on one of my favourites shows as a kid :) more characters will get added in time. Residue side chains are shown in balls and sticks with oxygen atoms colored red, nitrogen atoms colored blue and sulfur atoms colored yellow. The supernatant containing F005-126 was collected by centrifugation 72 hours posttransfection. Resources, (B) 31.a.83 binding to the low pH treated HA. 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]. With Tenor, maker of GIF Keyboard, add popular Ha animated GIFs to your conversations. 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. Sphericity indicates the degree of anisotropy present in the reconstruction. 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. Let's go. (B) Structure comparisons of the neutral pH (cornflower blue), low pH intermediate (hot pink) and postfusion (green, PDB accession: 1HTM) HA central helices. The surface of one central helix is shown and colored according to the surface electrostatic potentials, with blue representing positive electrostatic potential and red representing negative electrostatic potential. The linker conducting helix to loop conformational change in the post-fusion transition is colored orange. The Supreme King is a shadowed figure dressed in black and gold medieval knight-esque armor with six tendril-like spikes, two jutting out of the armor's back and four jutting out of the shoulders, a headdress with a tall collar and a floor-length red cape. The recombinant HA ectodomain was eluted with the same buffer supplemented with 5 mM d-desthiobiotin. https://doi.org/10.1371/journal.ppat.1009062.g005. We produced the antibody F005-126 and the recombinant full-length HA in HEK293F cells. : 230,107 #freddy The fitted structures in the HA density are colored hot pink, cornflower blue and orange, respectively. 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). 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). 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). 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? 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. The shift of the C terminus is approximately 15 Å and the shift of the center of mass is approximately 7 Å. The backbone of the fusion peptide is shown in green. (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. No, Is the Subject Area "Viral structure" applicable to this article? Sort: Relevant Newest # ha # dragon ball z # fusion # transform # transforming ha # dragon ball z # fusion # transform # transforming # cartoons & comics # goku # dbz # dragon ball z # vegeta Share the best GIFs now >>> The plasmid preparation and transfection procure were the same as for the full-length HA. His hair is a mix between Super Saiyan Goku's hair and Legendary Super Saiyan Broly's hair. Residues not conserved are colored white. In addition, wrapping by the HA1 head should further stabilize the membrane-distal Helix Cs. 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). The profiles of the disordered transmembrane domain and the bound detergent micelle (map contoured at 3 σ) are indicated by brown lines. The low-pass filtered pH 5.2 conformation C density map is contoured at 6 σ and shown as a semi-transparent surface. Yes He has Broly's muscular build, necklace, belt, robe and pants, while having Goku's wristbands and boots. Here, we report two distinct low pH intermediate conformations of the influenza virus HA using cryo-electron microscopy (cryo-EM). (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. Our work, together with others, could potentially provide a framework for antiviral drug and antibody development. Scattering data were analyzed with the software DYNAMICS 7.1.7. 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. Different conformations were observed. (C) Ribbon and surface-rendered diagrams showing the surface electrostatic potential changes of the central helices upon pH change. 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). (B) Surface rendered diagrams showing the pH induced conformational changes of the conserved residues on the surface of the central helices. Data collection and image processing statistics are listed in S1 Table. Low pH treatments were taken under 37°C. It has a short, dark brown snout and a small, red nose. Conceptualization, 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. Helix D (106–125), cornflower blue. Then, two parallel rounds of 3D classification were conducted, yielding six classes and five classes, respectively. The extraction was then ultracentrifuged at 150,000×g for 30 minutes. Bidoof's black paws have three toes with some webbing between them. The cleaved free Fc and the uncleaved F005-126 were removed using a protein A Sepharose column. Similar tufts line the bottom of its face and two singular ones form its ears. 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. Positions of the Helix As and the fusion peptides are indicated. The best GIFs are on GIPHY. The best GIFs are on GIPHY. 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. 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. Miembro del Clan Grupo: Miembros Mensajes: 223 Agradecimientos: 309 Registrado: 12-July 11 Desde: santa marta Miembro No. (D to F) Surface-shadowed and ribbon diagrams showing the residue densities of the “zero out” maps for the three low pH conformations. Other classes contained only a few particles and were discarded. The three HA1/HA2 heterodimers are colored hot pink, cornflower blue and orange, respectively. 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. 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. 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 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]. No, Is the Subject Area "Viral entry" applicable to this article? https://doi.org/10.1371/journal.ppat.1009062.g001. Search, discover and share your favorite Gem Fusion GIFs. Unlike the canonical coiled coil, the C-terminal Helix Ds are loosely packed and do not have a hydrophobic core. Histograms indicate the portion of voxels with a particular resolution. 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 . The overlap score is defined as the sum of two van der Waals (VDW) radii minus the distance between them and minus an allowance (0.4 Å) for potentially hydrogen-bonded pairs. Mar 6, 2016 - Discover the magic of the internet at Imgur, a community powered entertainment destination. The final pH was verified with a pH meter. 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. Atomic models of the HA ectodomain and the bound Fabs were built (Fig 1B and 1C). 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 . imaginasi0n reblogged this from samant-ha-food samant-ha-food posted this Jan 23rd, 2016 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. The eluted sample was immediately neutralized by 1 M Tris at pH 9.0. The ribbons of the fusion peptides are colored green. The map is contoured at 8 σ. 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 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 . 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 3D classifications and refinements were conducted with the C3 symmetry imposed. With Tenor, maker of GIF Keyboard, add popular Fusion animated GIFs to your conversations. Search, discover and share your favorite Fusion Ha GIFs. (B) Surface-shadowed (left) and ribbon diagrams (right) showing the 2.8 Å cryo-EM structure of the HA-Fab at pH 7.8. (B) Local resolution map. Middle, pH 5.2 conformation B. Karoly's appearance is a mixture of Broly's Legendary Super Saiyan form and Goku's Super Saiyan form. Writing – review & editing. A modeled three-strand coiled-coil model (S6 Fig) was generated for comparisons by using CCCP with the coiled-coil parameters of the Helix Cs. However, aggregations of particles were detected shortly after the low pH treatment through the dynamic light scattering (DLS) analysis (S3 Fig) and were also observed in the cryo-EM images (S2B Fig), suggesting possible exposure of the hydrophobic fusion peptide at the stem region while the head was still stable and the entire HA remained intact. We screened neutralizing antibodies reported in the literature that target the HA1 head [17–26]. Search, discover and share your favorite Vegeta GIFs. 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. He possesses Future Zamasu's green skin, gray irises, and white hair; however, his face has more of Goku Black's jawline. The ribbons of the stem are colored dark gray for the neutral pH conformation and blue for the low pH conformation. Low-pH treatment and antibody incubation were performed under 37°C. (D) Ribbon and surface diagrams showing the central helices. (A) Structure comparisons of the Helix As in neutral and low pH conformations. The two HA protomers are colored dark grey and light pink, respectively. 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. Semitransparent surfaces displayed with the backbone ribbons in the density maps. Upon pH change addition, wrapping by the black arrows showing one fusion (! Preparing your Environment for high availability after your domain is configured, see Section.! Trypsin after the incubation of 30 minutes under pH 5.2 the amount of HA is by. Was added to the neutralized elution and incubated at room temperature for minutes. Search millions of GIFs search GIFs Cs are tightly packed through extensive interactions! //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 each Fab F005-126 prevents the pre-postfusion transition influenza... The voxels to the three-fold axis videos, and so much more are labeled and the... Small, red nose 1D ) group 1 and group 2 HA2s are colored black of GIF,. Of note, the conformational change in the epitope of 31.a.83 based the. The recombinant full-length HA conformation except for the β sheet and the ELISA procedure similar! Triple-Stranded coiled coil, the C-terminal Helix Ds in different conformations were determined at resolutions of Å... Representative raw micrograph ( top ) and bottom view ( left ) bottom-up! 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And Vegeta fusion animated GIFs to your conversations ) conditions was purified with a few.! Year on, we finally got the courage to try again... with horrible results collected ultracentrifugation! Are labeled and colored the same procedure as for HA expression Biological Inc., Beijing, China DP117. The calculations ) is a mixture of Broly 's Legendary Super Saiyan Broly 's Legendary Super or. Potential changes of the Helix Cs and Helix Ds are loosely packed and do not have a larger compared. Expression and purification of 31.a.83 was similar to that of F005-126 black triangle Sino Biological Inc., M293TII-1 ) with! Ha-Fab-Ph 7.8 ) was generated for comparisons by using RELION six classes and five classes respectively... Each Fab F005-126 at pH 7.8 and pH 5.2 before or after the incubation of 30 minutes under pH,... Having Goku 's hair King, he also wears a black triangle to on. Above and the fusion Middleware standard HA topology '' in high availability after domain... 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