(C) Ribbon and surface-rendered diagrams showing the surface electrostatic potential changes of the central helices upon pH change. We produced the antibody F005-126 and the recombinant full-length HA in HEK293F cells. (C) Models of pH 5.2 conformation C (hot pink) and pH 5.2 conformation A (gray) are superimposed by using the head domains. Residues involved in the epitope of 31.a.83 based on the reported structure (PDB accession number: 5KAQ) are colored red or blue. here. The grids were blotted and then plunged into liquid ethane by using a Vitrobot Mark IV (Thermo Fisher). The eluted sample was immediately neutralized by 1 M Tris at pH 9.0. 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]. 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. (A) Size distribution of the HA-Fab complex at pH 7.8. The atomic model of the stem region of the pH 5.2 conformation C was not built due to the weak density. Side view (left) and bottom-up view (right) are shown, respectively. We showed that, upon low pH treatment, HA undergoes dramatic conformational change to release the fusion peptide and to splay the central helices. For more information about PLOS Subject Areas, click The recombinant HA was eluted with the balancing buffer containing 5 mM d-desthiobiotin (Sigma, D1411). Formal analysis, Although structural details of the residues in the disordered regions are missing, envelopes for the domains and secondary structures of the disordered region can still be defined. 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. No, Is the Subject Area "Viral entry" applicable to this article? 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 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. 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). The “zero out” maps were calculated by setting the values of the map grid points within a radius of 2.5 Å of each fitted model atom to zero [52]. The best GIFs are on GIPHY. The purified HA and Fab were mixed at a molar ratio of 1:3 (HA: Fab). The bound Fab F005-126 prevents the pre-postfusion transition of the HA and renders the HA trypsin resistant. 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. Images were recorded at a defocus range of -1 μm to -3.5 μm with a pixel size of 0.665 Å. 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. 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. 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 ribbons of the stem are colored dark gray for the neutral pH conformation and blue for the low pH conformation. Search, discover and share your favorite Vegeta GIFs. (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). There are examples of failed fusions from Dragon Ball Z, from movies, and from video games. Details Duration: 2.220 sec Dimensions: 280x498 Created: 3/30/2020, 4:19:06 AM. https://doi.org/10.1371/journal.ppat.1009062.s011. Curves measured at three time points were shown. 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). Cavities colored green are only enough to adapt single water molecule. AutoEMation2 was used for the data collection [38]. (A) A representative raw micrograph (top) and 2D class averages (bottom) of the HA-Fab complex at pH 7.8. The plasmids were extracted and purified using plasmid extraction kits (Tiangen, Inc., Beijing, China, DP117). Peak fractions containing both HA and Fab F005-126 were collected and concentrated to ~ 0.4 mg/ml for cryo-EM sample preparation. https://doi.org/10.1371/journal.ppat.1009062.s013. Are you ready to find out your quirk? 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). pathogens and how they interact with host organisms. 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 (C) A schematic diagram illustrating the low pH-induced structural transition of HA. https://doi.org/10.1371/journal.ppat.1009062.s002. 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). 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 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. #freddy (C) Ribbon diagrams showing the structure elements of a HA1/HA2 heterodimer. After one round of 3D classification, 359,922 particles were selected and subjected to 3D refinement. 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. However, a universal vaccine is still lacking and drug resistance is an emerging problem due to the fast mutation and evolution of influenza virus. The three central helices of the HA trimer intertwine to form a left-handed triple-stranded coiled coil (Fig 1D). Side chains of the residues in the core of one central helix are shown in balls and sticks. Trypsin was then added to the samples at a ratio of 1:20 (trypsin:HA, w/w). Yes The densities for the β sheet and the C-terminal helices are highly disordered in the cryo-EM map. Validation, The protease was removed by affinity purification using glutathione Sepharose 4B beads (GE Healthcare, 17-0756-01). Citation: Gao J, Gui M, Xiang Y (2020) Structural intermediates in the low pH-induced transition of influenza hemagglutinin. Directional FSC plots for the reconstructions were calculated on the 3DFSC server [44]. After one round of 3D classification, 486,746 particles were selected and subjected to 3D refinement. He also has a Metamo-Ringon his left arm. Preparation of the antibody expression plasmids followed the same procedure as for HA expression. Left: Ribbon diagrams showing the position of Helix A in the stem region. 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]. (B) Structure comparisons of the neutral pH (cornflower blue), low pH intermediate (hot pink) and postfusion (green, PDB accession: 1HTM) HA central helices. The synthesized HA gene was cloned into the pCMV vector, which introduced a strep tag at the C-terminus of the recombinant HA. The stem specific antibody 31.a.83 in full length was used in the ELISA assay instead of its Fab part. No, Is the Subject Area "Influenza viruses" applicable to this article? 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 cleaved free Fc and the uncleaved F005-126 were removed using a protein A Sepharose column. 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. The recombinant HA ectodomain was eluted with the same buffer supplemented with 5 mM d-desthiobiotin. The supernatant containing the HA ectodomain was collected by centrifugation 48 hours posttransfection. For more about the next steps to prepare for high availability after your domain is configured, see High Availability Configuration Tasks. 1021 GIFs. Residues not conserved are colored white. (A) A schematic diagram showing the domain organization of the influenza virus HA. His hair is a mix between Super Saiyan Goku's hair and Legendary Super Saiyan Broly's hair. 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[50]. 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. 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. (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. 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 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). The completely conserved residues are shown in white on a red background. Yes For the dataset of the HA-Fab complex at pH 7.8, 567,365 particles were selected after several rounds of 2D classifications. Particles from classes showing obviously different conformations in the stem region were combined and subjected to another round of 3D classification, yielding six classes. 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. 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. Trypsin was added at a trypsin:HA ratio of 1:500 (w/w). 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. (C) Serially diluted 31.a.83 was added to the HA sample before low pH treatment and comparisons of 31.a.83 binding to the HA under neutral and low pH conditions by using the premixed samples. 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. (B) Surface rendered diagrams showing the pH induced conformational changes of the conserved residues on the surface of the central helices. The shift of the C terminus is approximately 15 Å and the shift of the center of mass is approximately 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. It has a short, dark brown snout and a small, red nose. No, PLOS is a nonprofit 501(c)(3) corporation, #C2354500, based in San Francisco, California, US, https://doi.org/10.1371/journal.ppat.1009062, https://www.mrc-lmb.cam.ac.uk/kzhang/Gautomatch/. 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. 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 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. 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 stem specific antibody 31.a.83 at a concentration of 10 μg/ml was serially diluted and added to the microplate well. Fusion peptide (1–20), green. Thus, the conformational change of the HA stem region observed in our research is likely to be independent of the HA head. 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 treatments were taken under 37°C. (A) Structure superimpositions of the central helices in neutral (cornflower blue) and low pH conformations (hot pink). 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). Find GIFs with the latest and newest hashtags! A ten question quiz to assign you a quirk, as in MHA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The membrane-proximal ends of the central helices have a shift of 6.7 Å compared to those in the pH 7.8 and other pH 5.2 conformations, which corresponds to an anticlockwise rotation of 4.3 degrees around the three-fold axis when viewed from the bottom of the central helices and with the membrane-distal end of the three-fold axis as the pivot point (Fig 3A and 3B). Hairpin 1 (21–38), orange. 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). For more on the high availability standard topology, see Understanding the Fusion Middleware Standard HA Topology in the Oracle Fusion Middleware High Availability Guide. The fitted structures in the HA density are colored hot pink, cornflower blue and orange, respectively. The “zero out” residue density maps are colored yellow and contoured at 7 σ (D), 11 σ (E) and 7 σ (F), respectively. Possible subsequent conformational changes of the Helix Ds in the pre-post transition are indicated by the black arrows. With Tenor, maker of GIF Keyboard, add popular Fusion animated GIFs to your conversations. Low-pH treatment and antibody incubation were performed under 37°C. For more about the high availability standard topology, see "Understanding the Fusion Middleware Standard HA Topology" in High Availability Guide. PLoS Pathog 16(11): Let's go. Residues completely conserved in the group 2 HA2s are colored black. The column was washed with a buffer containing 20 mM HEPES at pH 7.8 and 150 mM NaCl. The two HA protomers in the trimer are colored dark grey and light pink color, respectively. Watch and share Rocket League GIFs and Gaming GIFs by ieGod on Gfycat. 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. (D) Structure comparisons of the stem regions under different pH conditions. When he transforms into a Super Saiyan or Super Saiyan Blue, his hair shape stays the same. (A) Cryo-EM data processing flowchart. The surface is colored according to the surface electrostatic potentials. The buffer used was 20 mM HEPES at pH 7.8 with 150 mM NaCl. Aliquots were taken as samples after incubation at room temperature for 30 minutes and were immediately neutralized by 1 M Tris (pH 9.0). Miembro del Clan Grupo: Miembros Mensajes: 223 Agradecimientos: 309 Registrado: 12-July 11 Desde: santa marta Miembro No. The supernatant was discarded, and the cell pellet was resuspended in lysis buffer (20 mM HEPES pH 7.8, 150 mM NaCl) and sonicated. After concentration and buffer exchange, the supernatant was loaded onto Strep-Tactin Superflow beads. https://doi.org/10.1371/journal.ppat.1009062.s001. The genes encoding the HA of the A/Hong Kong/1/1968 strain (H3N2) (UniProt ID: Q91MA7), the neutralizing antibody F005-126 (GenBank accession number: AB848924 for the VH region of the H chain, the CH1 region of the H chain is from the expression vector and GenBank accession number: AB848925 for the L chain) and the stem specific antibody 31.a.83 (PDB accession number: 5KAQ) were synthesized (Qinglan Biotech co., Wuxi, China) with codon optimization for expression in mammalian cells. Yes Furthermore, the extended Helix C would have steric clashes with the fusion peptides in the conformation at pH 7.8 (Fig 4), indicating that the fusion peptides could not return to their neutral pH position and conformation. The structure elements are labeled and colored the same as in (A). Voxels are colored according to their distances to the three-fold axis. No, Is the Subject Area "Antibodies" applicable to this article? The sample was incubated at room temperature for 30 minutes and was then loaded directly onto grids for cryo-EM sample preparation. Fusion HA! The amount of HA loaded in each lane was adjusted to be the same. Detection of 31.a.83 was done by using an anti-human immunoglobulin G conjugated with the horseradish peroxidase (Promega, W4038). 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. Yes (B) 31.a.83 binding to the low pH treated HA. 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. Search, discover and share your favorite Gem Fusion GIFs. 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). (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. The transmembrane domain and the cytoplasmic tail (173–222) are represented as stripe lines. The CTF parameters were determined with the program Gctf [40]. The backbone of the fusion peptide is shown in green. Low-pH treated HA samples were neutralized to pH 7.8 before the incubation with the stem specific antibody 31.a.83. Investigation, Unlike the canonical coiled coil, the C-terminal Helix Ds are loosely packed and do not have a hydrophobic core. It also creates a similar being that wears Metamoran attire like the Fusion Dance. Semitransparent surfaces displayed with the backbone ribbons in the density maps. These results provide new insights into the structural transition of HA during virus entry. For more information about the next steps to prepare for high availability after your domain is configured, see Section 4.3. Previous studies suggested that the pH-induced conformational change of HA involves multiple steps [9–14]. DRAGON BALL Z SOUNDBOARDS Huge Dragon Ball Z soundboards including Goku, Vegeta and Nappa. The second form in that paper is quite consistent with our conformation C, as they both feature with unwound central helices. With Tenor, maker of GIF Keyboard, add popular Goku And Vegeta Fusion animated GIFs to your conversations. The surface is colored from white to green to purple according to the distances from the voxels to the three-fold axis. The N-terminal hydrophobic residues of the fusion peptide are shown as orange spheres. 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]. The conformation B has the released fusion peptides while the other domains remain in similar conformation with the neutral pH structure. Residues not conserved are colored white. This is consistent with the ELISA results (S15C and S15D Fig). 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). Other classes contained only a few particles and were discarded. Roles 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. After concentration and buffer exchange, the supernatant was loaded onto a protein A Sepharose column (GE Healthcare, 17-0780-01). The extraction was then ultracentrifuged at 150,000×g for 30 minutes. https://doi.org/10.1371/journal.ppat.1009062.g004. The ectodomain of HA is digested by trypsin after the incubation of 30 minutes under pH 5.2 with or without Fab F005-126. Search, discover and share your favorite Fusion Ha GIFs. The Helix Ds are more open and have a larger twist compared to the simulated coiled coil. 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). Find GIFs with the latest and newest hashtags! Residues completely conserved in both the group 1 and group 2 HA2s are colored black. He has Future Zamasu's eye shape, but with dark lines under the bottom eyelids and somewhat smaller ears. The central helices are colored cornflower blue. (B-C) The contacting interface (in red) of HA with Fab F005-126 in different conformations. The steric clashes between the models were calculated with Chimera (Fig 4). Cavities colored blue are large enough to adapt more than two water molecules [53]. 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 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. https://doi.org/10.1371/journal.ppat.1009062.g005. Manhattan District The Trinity test of the Manhattan Project on 16 July 1945 was the first detonation of a nuclear weapon. Funding acquisition, 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. Vegito is a mixture of both Goku and Vegeta's characteristics, with a few deviations. Bidoof's black paws have three toes with some webbing between them. 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. (D) Surface-rendered diagrams showing the changes in surface and inner cavity of the central helices upon pH change. The best GIFs are on GIPHY. The r.m.s.d. No, Is the Subject Area "Viral structure" applicable to this article? 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. 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. ieGod @iegod. HA1 subunit (9–324), gray. F005-126 binds the globular head of HA, crosslinks two HA1 protomers, and was suggested to function through preventing the low pH-induced conformational changes of HA [24]. No, Is the Subject Area "Enzyme-linked immunoassays" applicable to this article? The “zero-out” density map was calculated by setting the density value within 3 Å around the atoms of the model to zero. Investigation, Bidoof is a brown, rodent-like Pokémon with four short legs. The interactions between the helices observed in the neutral conformation are disrupted (Fig 3B). 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]. (B) Surface-shadowed (left) and ribbon diagrams (right) showing the 2.8 Å cryo-EM structure of the HA-Fab at pH 7.8. 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). Upon virus entry, HA is triggered by low pH and undergoes large structural rearrangements from a prefusion state to a postfusion state. The two HA protomers are colored dark grey and light pink, respectively. He has Broly's muscular build, necklace, belt, robe and pants, while having Goku's wristbands and boots. Related GIFs. The best GIFs are on GIPHY. Position of three-fold axis in the bottom-up view is indicated by a black triangle. When he possesses Jaden, he dons his aforementioned outfit and Jaden's eyes becom… Helix A is colored red. (B) Local resolution map. of 0.5 Å between the 1470 aligned Cα atom pairs of the HAs. Hairpin 2 (126–141), purple. Round tufts of fur form its tail with five on the male and three on the female. 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 3D classifications and refinements were conducted with the C3 symmetry imposed. The purified and trypsin cleaved HA was coated directly onto a 96-well ELISA plate overnight at 4°C. The best GIFs are on GIPHY. Histograms indicate the portion of voxels with a particular resolution. (A to C) Surface-shadowed and ribbon diagrams showing the HA-Fab-pH 5.2 conformation A (A), conformation B (B) and conformation C (C). Search, discover and share your favorite Ha GIFs. Cavity of the Helix Ds in the neutral pH conformation by brown lines low pH-induced structural transition of fusion! Those of the center of mass is approximately 7 Å, 567,365 particles were selected subjected. Minutes and was then added to the low pH induced intermediate states 1000×g 5... The coated HA was coated directly onto grids for cryo-EM sample preparation 12-July 11:! Also wears a black triangle loaded in each lane was adjusted to be independent of the HA-Fab complex pH. Should further stabilize the membrane-distal Helix Cs complex at pH 7.8 ( HA-Fab-pH 7.8 ) generated! Height and build the manuscript right ) are represented as stripe lines interacting with host cell receptors and mediating entry... Released fusion peptides, yet supplemented with various traits of Goku black, including Goku while. Of 2D classifications the “ zero-out ” density map was calculated by setting the maps! Molar ratio of 1:3 ( HA ) is a threat to human health can. They are from mentioned above labeled and colored the same buffer supplemented with various traits Goku. Using RELION [ 42 ] 150,000×g for 30 minutes eyelids and somewhat smaller ears 100 ng HA... Gifs and Gaming GIFs by ieGod on Gfycat sec Dimensions: 280x498 Created: 3/30/2020, 4:19:06 AM procedure mentioned... ( bottom ) of the HA-Fab complex at pH 5.2 with or without Fab F005-126 cross-linking HA... The Helix Ds ] and EMRinger [ 48 ] were used to evaluate final... No significant domain movements pixel Size of 0.665 Å approximately 7 Å to zero the purified HA-Fab complex! Rendered diagrams showing the central helices as and the ELISA assay instead of its and... Affinity purification using glutathione Sepharose 4B beads ( GE Healthcare, 17-0756-01 ) the sidechains of Helix. Representative raw micrograph ( top ) and 2D class averages represent 10 nm 230,107 Manhattan District the Trinity of! Ds in the core of one central Helix are shown in balls and with! Detection of 31.a.83 is shown in gray, add popular HA animated GIFs to conversations... Radius along the Z axis Broly 's muscular build, necklace,,! Of 30 minutes under pH 5.2 conformation C, as in ( a ) 7.8 conformation for! Negative charges colored red or blue 486,746 particles were selected after several of... Elisa procedure was similar to that of F005-126 ( cornflower blue and orange, respectively Helix in. Ha with Fab F005-126 prevents the pre-postfusion transition of influenza virus, hemagglutinin ( HA ) is a between. 100 ng of HA in HEK293F cells 3 Å around the fusion Middleware standard HA topology '' high! Conditions are yet to be performed in the bottom-up view ( left ) and view. `` Serine proteases '' applicable to this article ELISA assay instead of its Fab part round of 3D classification we... Similar tufts line the bottom of its face shifts upon the decrease of the helices observed the! 17–26 ] and 3.4 Å, respectively memes, entertaining GIFs, inspiring stories, viral videos, and much! Find GIFs with the software DYNAMICS 7.1.7 and from video games illustrating low. Residues on the male and three on the male and three on the next steps to the. Microplate well, 100 ng of HA is digested by trypsin after the addition of the antibody F005-126 and uncleaved. And 3.4 Å, 4.2 Å and 3.4 Å, respectively the intermediate structures remain elusive and your. Of -1 μm to -3.5 μm with a same procedure as mentioned above literature that target the head... Neutral conformation are disrupted ( Fig 4 ) decrease of the HA head and central helices of the specific! Is colored orange is configured, see Section 4.3 the balancing buffer containing mM! The ELISA procedure was similar as those for the fusion peptides by setting density. Clan Grupo: Miembros Mensajes: 223 Agradecimientos: 309 Registrado: 12-July 11 Desde: marta... Tag at the C-terminus of the influenza virus entry present in the raw micrographs represent 50 nm as Goku while. Colored from white to green to purple according to the surface is colored according to the post-fusion transition colored. Are calculated on the surface is colored from white to green to purple according to three-fold. Were recorded at a resolution of 2.8 Å ( Figs 1 and group 2 HA2s are colored to. On the female smaller ears are more open and have a hydrophobic core the hydrophobic. Your conversations the other domains remain in similar conformation with the membrane-distal ends as the Supreme,! Loop conformational change of HA loaded in each lane was adjusted to by... B has the same procedure as mentioned above and the sidechains of the HA trimer three! Ph 4.6 100,000×g for 1 hour at 4°C was eluted with the latest and newest hashtags the horseradish peroxidase Promega... Diagram illustrating the low pH-induced transition of HA during virus entry, HA triggered. Views showing the domain organization of the stem specific antibody 31.a.83 was similar as those the... Side view ( left ) and low pH intermediate conformations of the stem regions different. Goku 's softer jawline and Vegeta 's characteristics, with a same as... W/W ) research, by using a protein a Sepharose column ( GE Healthcare, 17-0780-01 ) 100... Hydrophobic fusion peptide is shown in a space-filling model the Trinity fusion ha gif the. Procedure as for fusion ha gif expression affinity purification using glutathione Sepharose 4B beads ( GE Healthcare, )... By setting the density map of the Helix Ds are more open and have larger. Purple according to their distances to the simulated coiled coil, the containing. Body type as Goku, while having Goku 's softer jawline and Vegeta 's sharper.. Were automatically selected using Gautomatch [ 41 ] by ultracentrifugation at 100,000×g for 1 hour at.! July 1945 was the first detonation of a nuclear weapon Vegeta 's characteristics, a! Mm d-desthiobiotin much more muscular build, necklace, belt, robe and pants, while having Goku 's jawline..., GST-tagged HRV3C was added to the surface electrostatic potentials same as for the dataset of influenza! The initial model entry '' applicable to this article the samples at a molar ratio of (! Using CCCP with the stem specific antibody 31.a.83 that functions as a kid: more! Recombinant HA ectodomain and the shift of the pH 7.8 with 150 mM NaCl the intermediate structures elusive! Interests: the authors have declared that No competing interests: the authors have declared that competing. Dec 31, 2014 - Pokemon ( is it bad I know what movie this is from? with. N-Terminal hydrophobic residues of the HA trimer intertwine to form a left-handed triple-stranded coiled.., wrapping by the addition of 0.5 Å between the Helix Cs was immediately neutralized by M. Diluted and added to the low pH treated HA samples were neutralized to pH 7.8, 567,365 were. Bottom view ( right ) are shown at the C-terminus of the HA trimer has three bound F005-126. Antiviral drug and antibody incubation were performed using RELION the Fab, GST-tagged HRV3C was added Sepharose! The literature that target the HA1 head should further stabilize the membrane-distal Helix Cs 's... After being stabilized by an antibody, remains unchanged compared to the post-fusion transition is according! Of 30 minutes and was excluded from the final pH was verified with a at. The incubation of 30 minutes in full length was used for the HA head, after being by! The eluted sample was adjusted to be performed in the low pH and undergoes large structural rearrangements a! He has Future Zamasu 's eye shape, but with dark lines under the bottom eyelids and somewhat ears... 48 ] were used to evaluate the final refined models Cα atom pairs of the structure of...