|Entry||Database: EMDB / ID: EMD-5731|
|Title||Antibody Recognition of the Pandemic H1N1 Influenza Hemagglutinin Receptor Binding Site|
|Sample||Influenza hemagglutinin (A/Singapore/6/1986, H1N1) bound to neutralizing antibody (Fab 5J8)|
|Keywords||influenza / hemagglutinin / antibody / neutralizing / Fab|
|Biological species||Homo sapiens (human) / Influenza A virus (Flu)|
|Method||single particle reconstruction / negative staining / Resolution: 22 Å|
|Authors||Hong M / Lee PS / Hoffman RMB / Zhu X / Krause JC / Laursen NS / Yoon S / Song L / Tussey L / Crowe Jr JE / Ward AB / Wilson IA|
|Citation||Journal: J. Virol. / Year: 2013|
Title: Antibody recognition of the pandemic H1N1 Influenza virus hemagglutinin receptor binding site.
Authors: Minsun Hong / Peter S Lee / Ryan M B Hoffman / Xueyong Zhu / Jens C Krause / Nick S Laursen / Sung-Il Yoon / Langzhou Song / Lynda Tussey / James E Crowe / Andrew B Ward / Ian A Wilson
Abstract: Influenza virus is a global health concern due to its unpredictable pandemic potential. This potential threat was realized in 2009 when an H1N1 virus emerged that resembled the 1918 virus in ...Influenza virus is a global health concern due to its unpredictable pandemic potential. This potential threat was realized in 2009 when an H1N1 virus emerged that resembled the 1918 virus in antigenicity but fortunately was not nearly as deadly. 5J8 is a human antibody that potently neutralizes a broad spectrum of H1N1 viruses, including the 1918 and 2009 pandemic viruses. Here, we present the crystal structure of 5J8 Fab in complex with a bacterially expressed and refolded globular head domain from the hemagglutinin (HA) of the A/California/07/2009 (H1N1) pandemic virus. 5J8 recognizes a conserved epitope in and around the receptor binding site (RBS), and its HCDR3 closely mimics interactions of the sialic acid receptor. Electron microscopy (EM) reconstructions of 5J8 Fab in complex with an HA trimer from a 1986 H1 strain and with an engineered stabilized HA trimer from the 2009 H1 pandemic virus showed a similar mode of binding. As for other characterized RBS-targeted antibodies, 5J8 uses avidity to extend its breadth and affinity against divergent H1 strains. 5J8 selectively interacts with HA insertion residue 133a, which is conserved in pandemic H1 strains and has precluded binding of other RBS-targeted antibodies. Thus, the RBS of divergent HAs is targeted by 5J8 and adds to the growing arsenal of common recognition motifs for design of therapeutics and vaccines. Moreover, consistent with previous studies, the bacterially expressed H1 HA properly refolds, retaining its antigenic structure, and presents a low-cost and rapid alternative for engineering and manufacturing candidate flu vaccines.
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_5731.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 2.65 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire Influenza hemagglutinin (A/Singapore/6/1986, H1N1) bound to neutr...
|Entire||Name: Influenza hemagglutinin (A/Singapore/6/1986, H1N1) bound to neutralizing antibody (Fab 5J8)|
Number of components: 2 / Oligomeric State: One HA trimer bound to three Fabs
|Mass||Theoretical: 289 kDa|
-Component #1: protein, 5J8 Fab fragment
|Protein||Name: 5J8 Fab fragment / Oligomeric Details: monomer / Number of Copies: 3 / Recombinant expression: Yes|
|Mass||Theoretical: 46 kDa|
|Source||Species: Homo sapiens (human)|
|Source (engineered)||Expression System: Trichoplusia ni (cabbage looper) / Vector: pFastBac Dual / Cell of expression system: High Five|
-Component #2: protein, Influenza A/Singapore/6/1986 (H1N1) hemagglutinin
|Protein||Name: Influenza A/Singapore/6/1986 (H1N1) hemagglutinin / Oligomeric Details: trimer / Recombinant expression: Yes / Number of Copies: 1|
|Mass||Theoretical: 151 kDa|
|Source||Species: Influenza A virus (Flu) / Strain: A/Singapore/6/1986 (H1N1)|
|Source (engineered)||Expression System: Escherichia coli (E. coli) / Vector: pET24a|
|Specimen||Specimen state: Particle / Method: negative staining|
|Sample solution||Specimen conc.: 0.02 mg/mL / Buffer solution: 150 mM NaCl, 50 mM Tris / pH: 7.4|
|Support film||400 mesh copper grids coated in nitrocellulose and thin carbon, glow discharged in argon/oxygen|
|Staining||two cycles of "Nano-W" (2% methylamine tungstate) applied for 20 seconds|
|Vitrification||Cryogen name: NONE|
-Electron microscopy imaging
|Imaging||Microscope: FEI TECNAI 12 / Date: Feb 14, 2013|
|Electron gun||Electron source: LAB6 / Accelerating voltage: 120 kV / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 52000 X (nominal), 52000 X (calibrated)|
Astigmatism: Objective lens astigmatism corrected at 100,000 times magnification.
Imaging mode: BRIGHT FIELD / Defocus: 1300 nm
|Specimen Holder||Model: SIDE ENTRY, EUCENTRIC / Tilt Angle: 0 - 55 ° / Temperature: 298|
|Camera||Detector: TVIPS TEMCAM-F416 (4k x 4k)|
|Image acquisition||Number of digital images: 306 |
Details: Tilt series varying from 0-55 degrees, alternating low and high tilts on similarly stained regions
|Processing||Method: single particle reconstruction / Applied symmetry: C3 (3 fold cyclic) / Number of projections: 5106 |
Details: Particles were selected using automatic (difference-of-Gaussians) picking followed by reference-free classification to eliminate noisy picks or non-target aggregation states.
|3D reconstruction||Algorithm: Projection matching initiated with low-pass filtered hemagglutinin|
Euler angles: Eman1: 5 degrees / Software: Appion, Spider, Xmipp, Eman1 / Resolution: 22 Å
Resolution method: FSC at 0.5 cut-off (FSC calculated with Eman1's eotest procedure.)
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