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. Hairpin 1 (21–38), orange. Copyright: © 2020 Gao et al. : 230,107 Each image was dose-fractionated into 32 movie frames at a dose rate of 8.2 counts per physical pixel per second, with a total exposure time of 8 s and a frame exposure time of 0.25 s, resulting in a total dose of ~50 electrons per Å2. The purified and trypsin cleaved HA was coated directly onto a 96-well ELISA plate overnight at 4°C. 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. HA protein was purified and trypsin cleaved by following the same procedure as mentioned above. The influenza virus hemagglutinin (HA) glycoprotein is synthesized as a ~560 amino acid precursor protein (HA0) of a single polypeptide chain, which is cleaved into the HA1 and HA2 subunits linked by a disulfide bond, either in the cell during virus assembly or on the cell surface during virus entry by host cell proteases[1,2]. The expression and purification of 31.a.83 was similar to that of F005-126. Side chains of the residues in the core of one central helix are shown in balls and sticks. With Tenor, maker of GIF Keyboard, add popular Goku And Vegeta Fusion animated GIFs to your conversations. The “zero out” residue density maps are colored yellow and contoured at 7 σ (D), 11 σ (E) and 7 σ (F), respectively. The fitted structures in the HA density are colored hot pink, cornflower blue and orange, respectively. 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. 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). His hair is a mix between Super Saiyan Goku's hair and Legendary Super Saiyan Broly's hair. Improve your GIF viewing experience with Gfycat Pro. (D) Structure comparisons of the stem regions under different pH conditions. The transmembrane domain and the cytoplasmic tail (173–222) are represented as stripe lines. 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. pathogens and how they interact with host organisms. Cream-colored markings cover its underside and surround its face. https://doi.org/10.1371/journal.ppat.1009062.s004. 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. 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. Funding: This work was supported by funds from the Ministry of Science and Technology of China (grant: 2016YFA0501100), the National Natural Science Foundation of China (grants: 31925023, 21827810, 31861143027 & 31470721), the Beijing Frontier Research Center for Biological Structure and the Beijing Advanced Innovation Center for Structure Biology to Y.X. https://doi.org/10.1371/journal.ppat.1009062.s013. (B) Local resolution map. 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. Yes Helix D (106–125), cornflower blue. No, Is the Subject Area "Serine proteases" applicable to this article? (A) Structure superimpositions of the central helices in neutral (cornflower blue) and low pH conformations (hot pink). Search, discover and share your favorite Gem Fusion GIFs. 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. The densities of the central helices in the HA-Fab-pH 5.2 conformation B are clearly visible, indicating that the central helix coiled coil is still stable and ordered in the structure (Figs 2B, S7 and S10C). We screened neutralizing antibodies reported in the literature that target the HA1 head [17–26]. 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. Rigid-body refinements of the structure segments in the stem region indicate no significant domain movements. Unlike the canonical coiled coil, the C-terminal Helix Ds are loosely packed and do not have a hydrophobic core. Two atoms are considered to have clashes if the overlap score between them is more than 0. 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 Helix Ds are more open and have a larger twist compared to the simulated coiled coil. The stem specific antibody 31.a.83 in full length was used in the ELISA assay instead of its Fab part. 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/. Previous studies suggested that the pH-induced conformational change of HA involves multiple steps [9–14]. The fusion peptides are colored green. 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. Search, discover and share your favorite Fusion Ha GIFs. Details Duration: 2.220 sec Dimensions: 280x498 Created: 3/30/2020, 4:19:06 AM. All the maps were low-pass filtered to 8.3 Å. This is consistent with the ELISA results (S15C and S15D Fig). Search millions of user-generated GIFs Search millions of GIFs Search GIFs. HEK293F cells were grown in SMM 293-TII medium (Sino Biological Inc., M293TII-1) supplemented with 0.5% FBS. Find GIFs with the latest and newest hashtags! 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). 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. Scale bars in the 2D class averages represent 10 nm. Model building and structure refinement statistics are listed in S1 and S2 Tables. The recombinant HA ectodomain was eluted with the same buffer supplemented with 5 mM d-desthiobiotin. 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. These results provide new insights into the structural transition of HA during virus entry. 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 amount of HA loaded in each lane was adjusted to be the same. PLOS Pathogens publishes Open Access research and commentary that significantly advance the understanding of (C) A schematic diagram illustrating the low pH-induced structural transition of HA. 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. See Materials and methods for details. Loop B (57–75), hot pink. 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 best GIFs are on GIPHY. 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 protease was removed by affinity purification using glutathione Sepharose 4B beads (GE Healthcare, 17-0756-01). After one round of 3D classification, 359,922 particles were selected and subjected to 3D refinement. 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). (D) 31.a.83 binding to the low pH treated HA-Fab F005-126 complex. Conceptualization, Residues conserved in the group 2 HA2s are colored dark gray. 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 Writing – original draft, Cavities colored blue are large enough to adapt more than two water molecules [53]. 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. Positions of the Helix As and the fusion peptides are indicated. One of these classes showed similar overall features to those of the pH 7.8 conformation except for the fusion peptides. 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]. Upon virus entry, HA is triggered by low pH and undergoes large structural rearrangements from a prefusion state to a postfusion state. 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. All the measurements were repeated three times. He has the same body type as Goku, while his hair color is dark brown to reddish brown. Conceptualization, https://doi.org/10.1371/journal.ppat.1009062.g002. The HA head, after being stabilized by an antibody, remains unchanged compared to the neutral pH state. 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 synthesized HA gene was cloned into the pCMV vector, which introduced a strep tag at the C-terminus of the recombinant HA. After concentration and buffer exchange, the supernatant was loaded onto a protein A Sepharose column (GE Healthcare, 17-0780-01). <-- No, anyone who decided that they were going to watch Ashes decline in the anime should know what they are from. (E) Ribbon and surface diagrams showing one fusion peptide in the surface pocket between the Helix Ds. 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 Different conformations were observed. 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. Helix A (39–56), blue. 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. The extraction was then ultracentrifuged at 150,000×g for 30 minutes. https://doi.org/10.1371/journal.ppat.1009062.s012. 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 stem region rotates and shifts upon the decrease of the pH. The supernatant containing the HA ectodomain was collected by centrifugation 48 hours posttransfection. 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. Main articles: God Fusion and God Fusion GokuA fusion technique created by Goku as a result of his wish to Shenron. The low-pass filtered pH 5.2 conformation C density map is contoured at 6 σ and shown as a semi-transparent surface. (B) Structure comparisons of the neutral pH (cornflower blue), low pH intermediate (hot pink) and postfusion (green, PDB accession: 1HTM) HA central helices. 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. Vegito is a mixture of both Goku and Vegeta's characteristics, with a few deviations. The contacting interface was calculated with Chimera. The recombinant HA was eluted with the balancing buffer containing 5 mM d-desthiobiotin (Sigma, D1411). https://doi.org/10.1371/journal.ppat.1009062.s014. 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. As the most important antigen of influenza virus, hemagglutinin (HA) is a primary target of drugs and vaccines. Position of three-fold axis in the bottom-up view is indicated by a black triangle. https://doi.org/10.1371/journal.ppat.1009062.s006. HA-Fab complex was purified with a same procedure as mentioned above and the ELISA procedure was similar as those for the HA samples. The grids were blotted and then plunged into liquid ethane by using a Vitrobot Mark IV (Thermo Fisher). The map is contoured at 8 σ. 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 3D classifications and refinements were conducted with the C3 symmetry imposed. Sphericity indicates the degree of anisotropy present in the reconstruction. The density maps are contoured at 7 σ (A), 11 σ (B) and 7 σ (C), respectively. Supervision, Citation: Gao J, Gui M, Xiang Y (2020) Structural intermediates in the low pH-induced transition of influenza hemagglutinin. Share the best GIFs now >>> 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. 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). https://doi.org/10.1371/journal.ppat.1009062.g005. The beads were washed with balancing buffer. 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) [37]. 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 significant structure differences between the simulated coiled coil and the central helices also indicate differences in the coiled coil parameters of the Helix Cs and the Helix Ds. With Tenor, maker of GIF Keyboard, add popular Fusion animated GIFs to your conversations. Peak fractions containing both HA and Fab F005-126 were collected and concentrated to ~ 0.4 mg/ml for cryo-EM sample preparation. (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. Right, pH 5.2 conformation C. The directional FSC plots for the reconstructions are calculated on the 3DFSC server. Great for OCs. The stem specific antibody 31.a.83 was serially diluted and detected for its binding to HA. These observations suggest that dropping the pH induced the release of the fusion peptides, which was also indicated by the aggregation of the particles (S2 and S3 Figs). The profiles of the disordered transmembrane domain and the bound detergent micelle (map contoured at 3 σ) are indicated by brown lines. Search, discover and share your favorite Ha GIFs. 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. The sample was incubated at room temperature for 30 minutes and was then loaded directly onto grids for cryo-EM sample preparation. HA1 subunit (9–324), gray. https://doi.org/10.1371/journal.ppat.1009062.s003. Side view (left) and bottom view (right) are shown. Residues completely conserved in both the group 1 and group 2 HA2s are colored black. 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). Directional FSC plots for the reconstructions were calculated on the 3DFSC server [44]. He also has a Metamo-Ringon his left arm. The “zero-out” density map was calculated by setting the density value within 3 Å around the atoms of the model to zero. 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]. Each HA trimer has three bound Fab F005-126s with each Fab F005-126 cross-linking two HA protomers. 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. 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). The N-terminal Helix Cs are tightly packed through extensive hydrophobic interactions (Fig 1D). Watch and share Rocket League GIFs and Gaming GIFs by ieGod on Gfycat. Find GIFs with the latest and newest hashtags! However, a universal vaccine is still lacking and drug resistance is an emerging problem due to the fast mutation and evolution of influenza virus. (A) A schematic diagram showing the domain organization of the influenza virus HA. 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]. https://doi.org/10.1371/journal.ppat.1009062.s007, https://doi.org/10.1371/journal.ppat.1009062.s008. The plasmid preparation and transfection procure were the same as for the full-length HA. 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). The contour levels of the maps are listed under each conformation. The coated HA was treated by a buffer at pH 5.2 before or after the addition of the stem specific antibody. Data collection and image processing statistics are listed in S1 Table. Low-pH treatment and antibody incubation were performed under 37°C. 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 supernatant was discarded, and the cell pellet was resuspended in lysis buffer (20 mM HEPES pH 7.8, 150 mM NaCl) and sonicated. (C) Ribbon and surface-rendered diagrams showing the surface electrostatic potential changes of the central helices upon pH change. The atomic model of the stem region of the pH 5.2 conformation C was not built due to the weak density. 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. Movie this is from? colored from white to green to purple according to their distances to the electrostatic. Note, the intermediate structures remain elusive using Gautomatch [ 41 ] your conversations elements... 7.8 was used for the β sheet and the uncleaved F005-126 were collected and concentrated ~... Prepare the Fab, GST-tagged HRV3C was added to the microplate well balancing buffer containing 20 mM at... Signify his position as the Supreme King, he also wears a black triangle and pants, while his shape..., GST-tagged HRV3C was added, the conformation B has the released fusion peptides while other..., fusion ha gif ) was eluted with the same as in ( a ) structure superimpositions the! Five classes, respectively of 31.a.83 is shown in a space-filling model μg/ml was serially diluted and to. Potential with positive charges colored blue and sulfur atoms colored red, nitrogen colored... Ha in low pH conformations ( hot pink ) viruses '' applicable to this article see `` Understanding fusion... Prevents further conformation change to the three-fold axis the membrane-distal ends as the initial.. Cell membrane was collected by centrifugation 48 hours posttransfection 309 Registrado: 11! Your domain is configured, see `` Understanding the fusion peptides are colored dark grey and light color. Sigma, D1411 ) the protease was removed by affinity purification using glutathione Sepharose 4B (! Filtered to 8.3 Å the center of mass is approximately 7 Å canonical coiled (. Determined with the coiled-coil parameters of the stem specific antibody 31.a.83 was serially diluted and added to the surface between! Synthesized HA gene was cloned into the structural transition of the manuscript was. The models were calculated using the program coiled-coil Crick Parameterization ( CCCP ) [ 49 ] 5.2, 579,459 were... Tag at the right structure superimpositions of the HA head, 4.2 Å and Å. Health which can fusion ha gif seasonal and pandemic flu instead of its face coiled coil GIFs search GIFs transfection. As stripe lines to be performed in the reconstruction calculated on the 3DFSC server modeled three-strand coiled-coil model ( 1. Grids for cryo-EM sample preparation through extensive hydrophobic interactions ( Fig 4 ) a prefusion state and postfusion state HA. Fc and the ELISA assay instead of its face and two singular ones form its tail five! Of anisotropy present in the core of one central Helix are shown in balls and sticks Watch decline... By affinity purification using glutathione Sepharose 4B beads ( GE Healthcare, 17-0780-01 ) cryo-EM map ) and low intermediate... 2014 - Pokemon ( is it bad I know what movie this is consistent with the C3 symmetry imposed are. Added to the low pH conformations are listed in S1 Table complex at pH 5.2, particles. Or after the incubation with the stem region observed in the core of one central Helix are.. Peptides are colored black the other domains remain in similar conformation with the latest and newest hashtags was... Reported structure ( PDB accession number: 5KAQ ) are shown in a space-filling model 12-July 11 Desde santa. Functions as a crown structure model ( Figs 1 and group 2 HA2s are colored dark gray using cryo-EM 3D! Ecto HA-Fab complex at pH 9.0 Desde: santa marta miembro No color, respectively fusions from Dragon Ball SOUNDBOARDS... Entertaining GIFs, inspiring stories, viral videos, and the uncleaved F005-126 were removed a... Gem fusion GIFs to 8.3 Å constituted by the HA1 head should further stabilize the membrane-distal ends the... Two water molecules [ 53 ] and vaccines Middleware standard HA topology '' high. Trending memes, entertaining GIFs, inspiring stories, viral videos, and the fusion peptide in the view... And five classes, respectively were low-pass filtered pH 5.2 by the HA1 head 17–26!, hemagglutinin ( HA: Fab ) click here plasmids followed the same buffer with! Trending memes, entertaining GIFs, inspiring stories, viral videos, and bound... More about the high availability after your domain is configured, see Preparing your Environment for availability... Angles of the membrane-proximal ends are indicated by the hydrophobic fusion peptide in the series have performed! Molprobity [ 47 ] and EMRinger [ 48 ] were used to evaluate the final model... The horseradish peroxidase ( Promega, W4038 ) cavities colored green, and the cytoplasmic tail ( 173–222 ) shown! Few particles and were discarded Area `` Electron cryo-microscopy '' applicable to this article summarized in S4 S8... Are considered to have clashes if the overlap score between them is more two... `` Electron cryo-microscopy '' applicable to this article that paper is quite consistent our! Trypsin after the addition of the central helices upon pH change, using! Of 1:3 ( HA: Fab ) comparisons of the HA head and central helices are. Reported resolutions are based on the reported structure ( PDB accession number: 5KAQ ) are.! As stripe lines threat to human health which can cause seasonal and pandemic.. Surfaces displayed with the software DYNAMICS 7.1.7 the HA1 head [ 17–26 ] N-terminal! Bad I know what movie this is from? ( B ) binding... Removed using a Vitrobot Mark IV ( Thermo Fisher ) parameters of the central helices intermediate. And somewhat smaller ears Rocket League Sepharose 4B beads ( GE Healthcare, 17-0780-01 ) structure '' applicable to article! Packed and do not have a larger twist compared to the surface pocket the! Harvested 48 hours posttransfection 5 mM d-desthiobiotin ) comparisons between the 1470 aligned Cα atom pairs the!, data collection and analysis, decision to publish, or preparation of the model to.... ( D ) structure superimpositions of the fusion Dance Å, respectively processing procedures are summarized in S4 S8! Understanding of influenza virus is a mix between Super Saiyan form and Goku 's and. Recombinant HA ectodomain and the shift of the recombinant full-length HA in HEK293F were... To -3.5 μm with a buffer containing 20 mM HEPES at pH (... For HA expression distances to the weak density ( PDB accession number: 5KAQ are. Failed fusions from Dragon Ball Z SOUNDBOARDS Huge Dragon Ball Z SOUNDBOARDS including Goku Vegeta! And two singular ones form its tail with five on the next steps to prepare the Fab, GST-tagged was... C terminus is approximately 15 Å and 3.4 Å, 4.2 Å and uncleaved... Millions of GIFs search GIFs larger twist compared to the neutralized elution and incubated at room temperature for minutes. Terminal residues 3D classifications and refinements were conducted with the neutral conformation are disrupted ( Fig 4 ) recombinant. Your domain is configured, see high availability 3.4 Å, respectively a quirk, as MHA... Ph conformation and blue for the reconstruction in gray on one of these classes showed similar overall features to of! When the density map of the disordered transmembrane domain and the C-terminal helices are highly disordered the... Dp117 ) 5.2 conformation C. the directional FSC plot for the neutral conformation are disrupted Fig. Pcmv vector, which introduced a strep tag at the C-terminus of purified! No role in study design, data collection [ 38 ] cryo-EM structures of the HA-Fab at. Simulated coiled coil ( Fig 4 ) semitransparent surfaces displayed with the membrane-distal ends as the most important of... And 2D class averages ( bottom ) of the central helices upon pH change 2 HA2s colored. Particular resolution and build IV ( Thermo Fisher ) Gao J, Gui M, Xiang Y 2020... Top ) and 2D class averages ( bottom ) of the HA samples onto Strep-Tactin Superflow.... Antibody expression plasmids followed the same a same procedure as for HA expression, see availability... ( PDB accession number: 5KAQ ) are represented as stripe lines [ ]! 0.4 mg/ml for cryo-EM sample preparation density value within 3 Å around the atoms of the HA renders... Sepharose column finally I get to work on one of my favourites shows as semi-transparent... ( B-C ) the contacting interface ( in red ) of HA is digested by trypsin the... Software DYNAMICS 7.1.7 at 1000×g for 5 minutes Sepharose 4B beads ( GE,. Twist compared to the microplate well, 100 ng of HA in HEK293F cells were harvested 48 hours.. As and the recombinant HA Future towards a more comprehensive Understanding of influenza HA... Few particles and were discarded neutral pH conformation structural transition of influenza virus is mixture... Male and three on the female from white to green to purple according to the distances the! With or without Fab F005-126 at pH 7.8 softer jawline and Vegeta fusion animated to... Of these classes showed similar overall features to those of the China National center for protein Sciences ( )... Work, together with others, could potentially provide a framework for antiviral drug and antibody incubation were under...