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- EMDB-2074: map of the complex of the HA7 Fab with avian flu virus hemaggluti... -

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Basic information

Entry
Database: EMDB / ID: EMD-2074
Titlemap of the complex of the HA7 Fab with avian flu virus hemagglutinin by negative staining EM
Map data3D reconstruction from negative staining EM images of the trimeric Fab/hemagglutinin complex
Sample
  • Sample: the complex of Fab Fragment of HA7 monoclonal antibody and avian influenza virus hemagglutinin
  • Protein or peptide: Fab Fragment of HA7 monoclonal antibody
  • Protein or peptide: Avian influenza virus hemagglutinin
KeywordsHA7 antibody / Fab / hemagglutinin / avian flu virus
Function / homologyHaemagglutinin, influenzavirus A/B
Function and homology information
Biological speciesMus musculus (house mouse) / unidentified influenza virus
Methodsingle particle reconstruction / negative staining / Resolution: 18.0 Å
AuthorsDu L / Jin L / Zhao G / Sun S / Li J / Li Y / Zheng B / Liddington CL / Zhou Y / Jiang S
CitationJournal: J Virol / Year: 2013
Title: Identification and structural characterization of a broadly neutralizing antibody targeting a novel conserved epitope on the influenza virus H5N1 hemagglutinin.
Authors: Lanying Du / Lei Jin / Guangyu Zhao / Shihui Sun / Junfeng Li / Hong Yu / Ye Li / Bo-Jian Zheng / Robert C Liddington / Yusen Zhou / Shibo Jiang /
Abstract: The unabated circulation of the highly pathogenic avian influenza A virus/H5N1 continues to be a serious threat to public health worldwide. Because of the high frequency of naturally occurring ...The unabated circulation of the highly pathogenic avian influenza A virus/H5N1 continues to be a serious threat to public health worldwide. Because of the high frequency of naturally occurring mutations, the emergence of H5N1 variants with high virulence has raised great concerns about the potential transmissibility of the virus in humans. Recent studies have shown that laboratory-mutated or reassortant H5N1 viruses could be efficiently transmitted among mammals, particularly ferrets, the best animal model for humans. Thus, it is critical to establish effective strategies to combat future H5N1 pandemics. In this study, we identified a broadly neutralizing monoclonal antibody (MAb), HA-7, that potently neutralized all tested strains of H5N1 covering clades 0, 1, 2.2, 2.3.4, and 2.3.2.1 and completely protected mice against lethal challenges of H5N1 viruses from clades 1 and 2.3.4. HA-7 specifically targeted the globular head of the H5N1 virus hemagglutinin (HA). Using electron microscopy technology with three-dimensional reconstruction (3D-EM), we discovered that HA-7 bound to a novel and highly conserved conformational epitope that was centered on residues 81 to 83 and 117 to 122 of HA1 (H5 numbering). We further demonstrated that HA-7 inhibited viral entry during postattachment events but not at the receptor-binding step, which is fully consistent with the 3D-EM result. Taken together, we propose that HA-7 could be humanized as an effective passive immunotherapeutic agent for antiviral stockpiling for future influenza pandemics caused by emerging unpredictable H5N1 strains. Our study also provides a sound foundation for the rational design of vaccines capable of inducing broad-spectrum immunity against H5N1.
History
DepositionApr 13, 2012-
Header (metadata) releaseMay 17, 2012-
Map releaseMay 1, 2013-
UpdateJul 24, 2013-
Current statusJul 24, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.8
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.8
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2074.png

Downloads & links

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Map

FileDownload / File: emd_2074.map.gz / Format: CCP4 / Size: 1.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction from negative staining EM images of the trimeric Fab/hemagglutinin complex
Voxel sizeX=Y=Z: 4.28 Å
Density
Contour LevelBy AUTHOR: 1.8 / Movie #1: 1.8
Minimum - Maximum-10.62073421 - 13.96391296
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-36-36-36
Dimensions727272
Spacing727272
CellA=B=C: 308.16 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.284.284.28
M x/y/z727272
origin x/y/z0.0000.0000.000
length x/y/z308.160308.160308.160
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ128128168
MAP C/R/S123
start NC/NR/NS-36-36-36
NC/NR/NS727272
D min/max/mean-10.62113.9640.000

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Supplemental data

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Sample components

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Entire : the complex of Fab Fragment of HA7 monoclonal antibody and avian ...

EntireName: the complex of Fab Fragment of HA7 monoclonal antibody and avian influenza virus hemagglutinin
Components
  • Sample: the complex of Fab Fragment of HA7 monoclonal antibody and avian influenza virus hemagglutinin
  • Protein or peptide: Fab Fragment of HA7 monoclonal antibody
  • Protein or peptide: Avian influenza virus hemagglutinin

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Supramolecule #1000: the complex of Fab Fragment of HA7 monoclonal antibody and avian ...

SupramoleculeName: the complex of Fab Fragment of HA7 monoclonal antibody and avian influenza virus hemagglutinin
type: sample / ID: 1000 / Details: The trimeric sample was monodisperse.
Oligomeric state: one trimeric hemagglutinin binds to three Fab
Number unique components: 2
Molecular weightExperimental: 370 KDa / Theoretical: 370 KDa / Method: based on protein sequences and SDS-PAGE

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Macromolecule #1: Fab Fragment of HA7 monoclonal antibody

MacromoleculeName: Fab Fragment of HA7 monoclonal antibody / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Oligomeric state: trimer / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: House Mouse
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)

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Macromolecule #2: Avian influenza virus hemagglutinin

MacromoleculeName: Avian influenza virus hemagglutinin / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Oligomeric state: trimer / Recombinant expression: Yes
Source (natural)Organism: unidentified influenza virus
SequenceInterPro: Haemagglutinin, influenzavirus A/B

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4 / Details: 50mM NaCl, 10mM Tris-HCL,1mM CaCl2, pH7.4
StainingType: NEGATIVE
Details: Grids with adsorbed protein floated on 2% w/v uranyl formate for 30 seconds
GridDetails: 300 mesh copper grid with carbon support, glow discharged in vacuumed air
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 70000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 3.6 µm / Nominal defocus min: 1.2 µm
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 70,000 times magnification.
DateAug 7, 2011
Image recordingCategory: CCD / Film or detector model: GENERIC TVIPS (4k x 4k) / Number real images: 53 / Average electron dose: 50 e/Å2 / Bits/pixel: 8
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 18.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN2 / Details: C3 symmetry was imposed in refinement / Number images used: 3172
DetailsImage processing was done using EMAN2.The particles were selected by a semi-automatic selection using e2boxer.

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