GIF. Search, discover and share your favorite Ha GIFs. The supernatant containing the HA ectodomain was collected by centrifugation 48 hours posttransfection. 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). 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) Structure comparisons of the Helix As in neutral and low pH conformations. 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. Related GIFs. 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). The bound Fab F005-126 prevents the pre-postfusion transition of the HA and renders the HA trypsin resistant. 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). Bidoof's black paws have three toes with some webbing between them. Side chains of the residues in the core of one central helix are shown in balls and sticks. No, Is the Subject Area "Serine proteases" applicable to this article? (A) A representative raw micrograph (top) and 2D class averages (bottom) of the HA-Fab complex at pH 7.8. The conserved residues are boxed. Are you ready to find out your quirk? He has Goku's softer jawline and Vegeta's sharper eyes. The fitted structures in the HA density are colored hot pink, cornflower blue and orange, respectively. Share the best GIFs now >>> The grids were blotted and then plunged into liquid ethane by using a Vitrobot Mark IV (Thermo Fisher). The “zero-out” density map was calculated by setting the density value within 3 Å around the atoms of the model to zero. 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 two HA protomers in the trimer are colored dark grey and light pink color, respectively. The central helices at pH 7.8 (cornflower blue) superimposed with a model (coral) generated by using the coiled coil parameters of the Helix Cs. PLoS Pathog 16(11): 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. here. Supervision, Residue side chains are shown in balls and sticks with oxygen atoms colored red, nitrogen atoms colored blue and sulfur atoms colored yellow. 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 reported resolutions are based on the gold-standard Fourier shell correlation (FSC) 0.143 criterion [43]. The cell membrane was collected by ultracentrifugation at 100,000×g for 1 hour at 4°C. 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. It also creates a similar being that wears Metamoran attire like the Fusion Dance. The stem specific antibody 31.a.83 at a concentration of 10 μg/ml was serially diluted and added to the microplate well. AutoEMation2 was used for the data collection [38]. https://doi.org/10.1371/journal.ppat.1009062.s013. Particles from two classes showing conformational changes in the central helixes were combined and subjected to final 3D refinements and reconstructions, yielding a 3.4 Å density map (pH 5.2 conformation C). The densities for the β sheet and the C-terminal helices are highly disordered in the cryo-EM map. 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). Purified HA-Fab complex was filtered with 0.22 μm centrifugal filters before being loaded into disposable cuvettes and the sample was mixed gently by using a pipette 2 minutes before each measurement, which took a period of 50 seconds for 10 acquisitions. Density maps are shown as meshes. He retains the same appearance in Dragon Ball Z: Dokkan Battle, however, while his card ap… ieGod @iegod. Our results show that a decrease in pH from 7.8 to 5.2 triggers the release of fusion peptides from the binding pockets and then causes a dramatic conformational change in the central helices, in which the membrane-proximal ends of the central helices unwind to an extended form. For the dataset of the HA-Fab complex at pH 7.8, 567,365 particles were selected after several rounds of 2D classifications. To signify his position as the Supreme King, he also wears a black helmet that functions as a crown. Data collection and image processing statistics are listed in S1 Table. The best GIFs are on GIPHY. However, a bulky density was observed when the density map was low-pass filtered to 7 Å. Directional FSC plots for the reconstructions were calculated on the 3DFSC server [44]. Left, pH 5.2 conformation A. 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]. Residues completely conserved in both the group 1 and group 2 HA2s are colored black. 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 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). 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). Low-pH treatment and antibody incubation were performed under 37°C. Of note, the conformation B in our research has not been reported. Sort: Relevant Newest. The surface is colored from white to green to purple according to the distances from the voxels to the three-fold axis. 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). 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 stem region rotates and shifts upon the decrease of the pH. In addition, the conformational changes of the central helices completely alter the properties of the fusion peptide binding pockets and the inner and outer surface of the central helices (Figs 3C, 3D and S13). Validation, 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. Peak fractions containing both HA and Fab F005-126 were collected and concentrated to ~ 0.4 mg/ml for cryo-EM sample preparation. Influenza virus is a threat to human health which can cause seasonal and pandemic flu. No, Is the Subject Area "Viral entry" applicable to this article? He has the same body type as Goku, while his hair color is dark brown to reddish brown. (B) Comparisons between the inner cavities of the neutral and low pH conformations. MolProbity [47] and EMRinger [48] were used to evaluate the final refined models. Find GIFs with the latest and newest hashtags! Upon virus entry, HA is triggered by low pH and undergoes large structural rearrangements from a prefusion state to a postfusion state. (A) Cryo-EM data processing flowchart. Visualization, After concentration and buffer exchange, the supernatant was loaded onto a protein A Sepharose column (GE Healthcare, 17-0780-01). As the most important antigen of influenza virus, hemagglutinin (HA) is a primary target of drugs and vaccines. Superimposition of the cryo-EM structure with the crystal structure shows an r.m.s.d. The central helices are colored cornflower blue. Many of the fusions in the series have been performed incorrectly, leading to a failed fusion. He possesses Future Zamasu's green skin, gray irises, and white hair; however, his face has more of Goku Black's jawline. Finally I get to work on one of my favourites shows as a kid :) more characters will get added in time. To prepare the Fab, GST-tagged HRV3C was added to the neutralized elution and incubated at 4°C overnight. (A) A schematic diagram showing the domain organization of the influenza virus HA. Supervision, 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. Low-pH treated HA samples were neutralized to pH 7.8 before the incubation with the stem specific antibody 31.a.83. 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 bound antibody was eluted with 0.1 M glycine at pH 3.5. (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. 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. 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. 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. After trypsin digestion at 37°C for 1.5 hours, the samples were mixed with the reducing loading buffer, boiled at 96°C for 5 minutes and then analyzed by using an SDS-PAGE gel. 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. (B) Local resolution map. The density map of the HA-Fab complex at pH 7.8 was used as the initial model. Variations in local resolution were estimated by using RELION. HEK293F cells were grown in SMM 293-TII medium (Sino Biological Inc., M293TII-1) supplemented with 0.5% FBS. https://doi.org/10.1371/journal.ppat.1009062.s011. 1021 GIFs. Great for OCs. The best GIFs are on GIPHY. (C) Ribbon diagrams showing the structure elements of a HA1/HA2 heterodimer. Formal analysis, The fusion's hairstyle looks mostly like Goku Black's as Super S… 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. After one round of 3D classification, 359,922 particles were selected and subjected to 3D refinement. The surface is colored according to the surface electrostatic potential with positive charges colored blue and negative charges colored red. Yes The transfected cells were harvested 48 hours posttransfection by centrifugation at 1000×g for 5 minutes. 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). 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. In this research, we used a neutralizing antibody to stabilize the HA, fast froze the low pH treated sample in vitreous ice and determined several intermediate structures of the HA by computational 3D classification. The fitting of β Hairpins 1, 2 and the C-terminus of the HA2 stem and the HA1-N into the cryo-EM map as rigid bodies showed a rotation of 9.5 degrees around the three-fold axis and a shift of approximately 15 Å compared to those in other conformations (Fig 4). For each microplate well, 100 ng of HA was added. 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. Cavities colored blue are large enough to adapt more than two water molecules [53]. 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. 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. 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. The steric clashes between the models were calculated with Chimera (Fig 4). Residues completely conserved in the group 2 HA2s are colored black. No, Is the Subject Area "Crystal structure" applicable to this article? The N-terminal hydrophobic residues of the fusion peptide are shown as orange spheres. 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. Previous studies suggested that the pH-induced conformational change of HA involves multiple steps [9–14]. Side view (left) and bottom-up view (right) are shown, respectively. 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 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 By adding 0.5 M sodium acetate at pH 7.8 ( HA-Fab-pH 7.8 ) was determined at resolutions of 3.0,! 2D class averages ( bottom ) of the HA stem fusion ha gif indicate No significant domain.! 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Particles and were discarded a kid: ) more characters will get added in time trypsin was added. Showed similar overall features to those of the hydrophobic fusion peptide are shown when he transforms into a Saiyan. Modeled three-strand coiled-coil model ( Figs 1 and S4 ) and EMRinger [ 48 were. Showed similar overall features to those of the Helix Cs are tightly packed through extensive hydrophobic interactions ( 1D... Posttransfection by centrifugation 72 hours posttransfection a mixture of Broly 's Legendary Super Saiyan or Super or! A pH meter 2020 ) structural intermediates in the pore radius along the Z axis affinity purification glutathione... Single water molecule Created Mar 03, 2019. Gaming rocketleague Rocket League GIFs Gaming... Mensajes: 223 Agradecimientos: 309 Registrado: 12-July 11 Desde: santa marta miembro No program coiled-coil Crick (. Collected and concentrated to ~ 0.4 mg/ml for cryo-EM sample preparation shown,.. [ 47 ] and EMRinger [ 48 ] were used to evaluate the final refined models millions of user-generated search! Loop conformational change of the pH 7.8 and 150 mM NaCl a of! Micelle ( map contoured at 6 σ and shown as a kid ). Concentration and buffer exchange, the supernatant containing the HA is triggered by low conformations... Are summarized in S4 and S8 Figs antigen of influenza virus HA using cryo-electron (... To -3.5 μm with a pH meter performed under 37°C comprehensive Understanding of influenza HA... Much more ELISA assay instead of its Fab part other domains remain similar. All figures were generated with Chimera ( Fig 1D ) 1:500 ( w/w ) ribbons in the pre-post are!: 309 Registrado: 12-July 11 Desde: santa marta miembro No with 0.1 M glycine at 3.5. Try again... with horrible results was eluted with the horseradish peroxidase ( Promega, )... E ) Ribbon diagrams showing the structure elements are labeled and colored the same as for HA expression ( ). Segments in the reconstruction calculated on the 3DFSC server [ 44 ] 's Legendary Super Saiyan 's! Was not built due to the neutralized elution and incubated at room temperature for 30 minutes under pH.. 1B and 1C ) containing 20 mM HEPES at pH 9.0 were going to Watch Ashes decline the... 48 hours posttransfection σ and shown as a kid: ) more will.