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- EMDB-5731: Antibody Recognition of the Pandemic H1N1 Influenza Hemagglutinin... -

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Basic information

Entry
Database: EMDB / ID: EMD-5731
TitleAntibody Recognition of the Pandemic H1N1 Influenza Hemagglutinin Receptor Binding Site
Map dataInfluenza hemagglutinin (A/Singapore/6/1986, H1N1) bound to neutralizing antibody (Fab 5J8)
Sample
  • Sample: Influenza hemagglutinin (A/Singapore/6/1986, H1N1) bound to neutralizing antibody (Fab 5J8)
  • Protein or peptide: 5J8 Fab fragment
  • Protein or peptide: Influenza A/Singapore/6/1986 (H1N1) hemagglutinin
Keywordsinfluenza / hemagglutinin / antibody / neutralizing / Fab
Biological speciesHomo sapiens (human) / Influenza A virus
Methodsingle particle reconstruction / negative staining / Resolution: 22.0 Å
AuthorsHong M / Lee PS / Hoffman RMB / Zhu X / Krause JC / Laursen NS / Yoon S / Song L / Tussey L / Crowe Jr JE ...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
CitationJournal: 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.
History
DepositionJul 29, 2013-
Header (metadata) releaseAug 28, 2013-
Map releaseSep 25, 2013-
UpdateOct 30, 2013-
Current statusOct 30, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.42
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 2.42
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5731_1.png

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Map

FileDownload / File: emd_5731.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationInfluenza hemagglutinin (A/Singapore/6/1986, H1N1) bound to neutralizing antibody (Fab 5J8)
Voxel sizeX=Y=Z: 2.65 Å
Density
Contour LevelBy AUTHOR: 2.42 / Movie #1: 2.42
Minimum - Maximum-4.31275845 - 14.250058170000001
Average (Standard dev.)0.00004023 (±0.84270173)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-24-24-24
Dimensions128128128
Spacing128128128
CellA=B=C: 339.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.652.652.65
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z339.200339.200339.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS-24-24-24
NC/NR/NS128128128
D min/max/mean-4.31314.2500.000

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Supplemental data

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Sample components

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Entire : Influenza hemagglutinin (A/Singapore/6/1986, H1N1) bound to neutr...

EntireName: Influenza hemagglutinin (A/Singapore/6/1986, H1N1) bound to neutralizing antibody (Fab 5J8)
Components
  • Sample: Influenza hemagglutinin (A/Singapore/6/1986, H1N1) bound to neutralizing antibody (Fab 5J8)
  • Protein or peptide: 5J8 Fab fragment
  • Protein or peptide: Influenza A/Singapore/6/1986 (H1N1) hemagglutinin

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Supramolecule #1000: Influenza hemagglutinin (A/Singapore/6/1986, H1N1) bound to neutr...

SupramoleculeName: Influenza hemagglutinin (A/Singapore/6/1986, H1N1) bound to neutralizing antibody (Fab 5J8)
type: sample / ID: 1000 / Oligomeric state: One HA trimer bound to three Fabs / Number unique components: 2
Molecular weightTheoretical: 289 KDa

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Macromolecule #1: 5J8 Fab fragment

MacromoleculeName: 5J8 Fab fragment / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 46 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant cell: High Five / Recombinant plasmid: pFastBac Dual

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Macromolecule #2: Influenza A/Singapore/6/1986 (H1N1) hemagglutinin

MacromoleculeName: Influenza A/Singapore/6/1986 (H1N1) hemagglutinin / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Oligomeric state: trimer / Recombinant expression: Yes
Source (natural)Organism: Influenza A virus / Strain: A/Singapore/6/1986 (H1N1) / synonym: Flu
Molecular weightTheoretical: 151 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET24a

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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

Concentration0.02 mg/mL
BufferpH: 7.4 / Details: 150 mM NaCl, 50 mM Tris
StainingType: NEGATIVE
Details: two cycles of "Nano-W" (2% methylamine tungstate) applied for 20 seconds
GridDetails: 400 mesh copper grids coated in nitrocellulose and thin carbon, glow discharged in argon/oxygen
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 52000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.3 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 52000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle max: 55
TemperatureAverage: 298 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism corrected at 100,000 times magnification.
DateFeb 14, 2013
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 306
Details: Tilt series varying from 0-55 degrees, alternating low and high tilts on similarly stained regions
Tilt angle min0

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Image processing

Final angle assignmentDetails: Eman1: 5 degrees
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 22.0 Å / Resolution method: OTHER / Software - Name: Appion, Spider, Xmipp, Eman1 / Number images used: 5106
DetailsParticles were selected using automatic (difference-of-Gaussians) picking followed by reference-free classification to eliminate noisy picks or non-target aggregation states.

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