Helix A (39–56), blue. Writing – review & editing, * E-mail: miao_gui@hms.harvard.edu (MG); yxiang@mail.tsinghua.edu.cn (YX), Current address: Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, Massachusetts, United States of America, Roles The antibody stabilizes the HA head and prevents further conformation change to the post-fusion state. 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. https://doi.org/10.1371/journal.ppat.1009062.s012. He has Broly's muscular build, necklace, belt, robe and pants, while having Goku's wristbands and boots. He retains the same appearance in Dragon Ball Z: Dokkan Battle, however, while his card ap… The reported resolutions are based on the gold-standard Fourier shell correlation (FSC) 0.143 criterion [43]. The best GIFs are on GIPHY. e1009062. HA protein was purified and trypsin cleaved by following the same procedure as mentioned above. Particles were automatically selected using Gautomatch [41]. One of these classes showed similar overall features to those of the pH 7.8 conformation except for the fusion peptides. Left, pH 5.2 conformation A. The conformation B has the released fusion peptides while the other domains remain in similar conformation with the neutral pH structure. For more about the high availability standard topology, see "Understanding the Fusion Middleware Standard HA Topology" in High Availability Guide. 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. 12.8K views | Created Mar 03, 2019. gaming rocketleague rocket league. 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 Accordingly, the conformation change in the central helices will affect the binding of stem specific antibodies as well, especially for these targeting around the Helix A. The purified and trypsin cleaved HA was coated directly onto a 96-well ELISA plate overnight at 4°C. Find GIFs with the latest and newest hashtags! (C) A schematic diagram illustrating the low pH-induced structural transition of HA. His hair is a mix between Super Saiyan Goku's hair and Legendary Super Saiyan Broly's hair. 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 stem specific antibody 31.a.83 was serially diluted and detected for its binding to HA. 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). (B-C) The contacting interface (in red) of HA with Fab F005-126 in different conformations. The N-terminal hydrophobic residues of the fusion peptide are shown as orange spheres. Supervision, 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. In addition, wrapping by the HA1 head should further stabilize the membrane-distal Helix Cs. Scale bars in the 2D class averages represent 10 nm. pathogens and how they interact with host organisms. Helix D (106–125), cornflower blue. The beads were washed with balancing buffer. 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]. 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]. 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. Manhattan District The Trinity test of the Manhattan Project on 16 July 1945 was the first detonation of a nuclear weapon. 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). Voxels are colored according to their distances to the three-fold axis. 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). 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. Yes 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. (A) Changes in the pore radius along the Z axis. Further studies under physiological conditions are yet to be performed in the future towards a more comprehensive understanding of influenza virus entry. Karoly's appearance is a mixture of Broly's Legendary Super Saiyan form and Goku's Super Saiyan form. of 0.5 Å between the 1470 aligned Cα atom pairs of the HAs. The HA head, after being stabilized by an antibody, remains unchanged compared to the neutral pH state. 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). Influenza virus is a threat to human health which can cause seasonal and pandemic flu. 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. 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. Possible subsequent conformational changes of the Helix Ds in the pre-post transition are indicated by the black arrows. The transmembrane domain of the HA is flexible and was excluded from the final structure model (Figs 1B and S4). Density maps are shown as meshes. 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. Dec 31, 2014 - Pokemon (Is it bad I know what movie this is from?) Variations in local resolution were estimated by using RELION. The head encompasses the sialic acid receptor binding domains (RBDs), while the stem has a hydrophobic fusion peptide at the N-terminus and the transmembrane helix at the C-terminus of each HA2. (B) A representative raw micrograph (top) and 2D class averages (bottom) of the HA-Fab complex at pH 5.2. Middle, pH 5.2 conformation B. Superimposition of the cryo-EM structure with the crystal structure shows an r.m.s.d. The ectodomain of HA is digested by trypsin after the incubation of 30 minutes under pH 5.2 with or without Fab F005-126. Round tufts of fur form its tail with five on the male and three on the female. 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. For more about the next steps to prepare for high availability after your domain is configured, see High Availability Configuration Tasks. Get started. Data collection and image processing statistics are listed in S1 Table. Funding acquisition, Upload Create. 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. With Tenor, maker of GIF Keyboard, add popular Ha animated GIFs to your conversations. (D) Surface-rendered diagrams showing the changes in surface and inner cavity of the central helices upon pH change. 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 density maps are contoured at 7 σ (A), 11 σ (B) and 7 σ (C), respectively. Investigation, 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. 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. https://doi.org/10.1371/journal.ppat.1009062.s015, https://doi.org/10.1371/journal.ppat.1009062.s016, https://doi.org/10.1371/journal.ppat.1009062.s017. We produced the antibody F005-126 and the recombinant full-length HA in HEK293F cells. Is the Subject Area "Electron cryo-microscopy" applicable to this article? https://doi.org/10.1371/journal.ppat.1009062.s014. The CTF parameters were determined with the program Gctf [40]. Here, we report two distinct low pH intermediate conformations of the influenza virus HA using cryo-electron microscopy (cryo-EM). 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). 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. Mar 6, 2016 - Discover the magic of the internet at Imgur, a community powered entertainment destination. After concentration and buffer exchange, the supernatant was loaded onto Strep-Tactin Superflow beads. PLOS Pathogens publishes Open Access research and commentary that significantly advance the understanding of Lift your spirits with funny jokes, trending memes, entertaining gifs, inspiring stories, viral videos, and so much more. The low pH sample is in a buffer containing 150 mM NaCl, 20 mM HEPES at pH 5.2 and 0.003% LMNG and the complex is at a concentration of 0.07 mg/ml. Zoom-in views of the fitted region are shown at the right. The r.m.s.d. 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. Main articles: God Fusion and God Fusion GokuA fusion technique created by Goku as a result of his wish to Shenron. We screened neutralizing antibodies reported in the literature that target the HA1 head [17–26]. 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). https://doi.org/10.1371/journal.ppat.1009062.s013. The surface is colored according to the surface electrostatic potential with positive charges colored blue and negative charges colored red. The fusion peptide wraps around the N-terminal fragment of HA1 (residues 9–19 of HA1, HA1-N) and forms a hydrogen bond with residue His17 of HA1-N. Hydrophilic pockets are formed between the Helix Ds (Fig 1D and 1E). 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 funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Each HA trimer has three bound Fab F005-126s with each Fab F005-126 cross-linking two HA protomers. 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