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

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Database: EMDB / ID: EMD-5731
TitleAntibody Recognition of the Pandemic H1N1 Influenza Hemagglutinin Receptor Binding Site
Map data
SampleInfluenza hemagglutinin (A/Singapore/6/1986, H1N1) bound to neutralizing antibody (Fab 5J8)
  • 5J8 Fab fragment
  • Influenza A/Singapore/6/1986 (H1N1) hemagglutinin
Keywordsinfluenza / hemagglutinin / antibody / neutralizing / Fab
Biological speciesHomo sapiens (human) / Influenza A virus (Flu)
Methodsingle particle reconstruction / negative staining / Resolution: 22 Å
AuthorsHong 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.
Current status-Processing site: RCSB / Status: Released
DepositionJul 29, 2013-
Header (metadata) releaseAug 28, 2013-
Map releaseSep 25, 2013-
UpdateOct 30, 2013-

Structure visualization

  • 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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Structure viewerEM map:
Supplemental images

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FileDownload / File: emd_5731.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

AxesZ (Sec.)Y (Row.)X (Col.)
2.65 Å/pix.
x 128 pix.
= 339.2 Å
2.65 Å/pix.
x 128 pix.
= 339.2 Å
2.65 Å/pix.
x 128 pix.
= 339.2 Å



Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.65 Å
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


Map geometry
Axis orderXYZ
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
start NX/NY/NZ-132-122-147
MAP C/R/S123
start NC/NR/NS-24-24-24
D min/max/mean-4.31314.2500.000

Supplemental data

Sample components

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)
Number of components: 2 / Oligomeric State: One HA trimer bound to three Fabs
MassTheoretical: 289 kDa

Component #1: protein, 5J8 Fab fragment

ProteinName: 5J8 Fab fragment / Oligomeric Details: monomer / Number of Copies: 3 / Recombinant expression: Yes
MassTheoretical: 46 kDa
SourceSpecies: 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

ProteinName: Influenza A/Singapore/6/1986 (H1N1) hemagglutinin / Oligomeric Details: trimer / Recombinant expression: Yes / Number of Copies: 1
MassTheoretical: 151 kDa
SourceSpecies: Influenza A virus (Flu) / Strain: A/Singapore/6/1986 (H1N1)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pET24a

Experimental details

Sample preparation

SpecimenSpecimen state: Particle / Method: negative staining
Sample solutionSpecimen conc.: 0.02 mg/mL / Buffer solution: 150 mM NaCl, 50 mM Tris / pH: 7.4
Support film400 mesh copper grids coated in nitrocellulose and thin carbon, glow discharged in argon/oxygen
Stainingtwo cycles of "Nano-W" (2% methylamine tungstate) applied for 20 seconds
VitrificationCryogen name: NONE

Electron microscopy imaging

ImagingMicroscope: FEI TECNAI 12 / Date: Feb 14, 2013
Electron gunElectron source: LAB6 / Accelerating voltage: 120 kV / Illumination mode: FLOOD BEAM
LensMagnification: 52000 X (nominal), 52000 X (calibrated)
Astigmatism: Objective lens astigmatism corrected at 100,000 times magnification.
Imaging mode: BRIGHT FIELD / Defocus: 1300 nm
Specimen HolderModel: SIDE ENTRY, EUCENTRIC / Tilt Angle: 0 - 55 ° / Temperature: 298
CameraDetector: TVIPS TEMCAM-F416 (4k x 4k)

Image acquisition

Image acquisitionNumber of digital images: 306
Details: Tilt series varying from 0-55 degrees, alternating low and high tilts on similarly stained regions

Image processing

ProcessingMethod: 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 reconstructionAlgorithm: 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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