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- EMDB-6613: Cryo-electron microscopy structure of chimeric influenza cH5/1 he... -

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Basic information

Entry
Database: EMDB / ID: EMD-6613
TitleCryo-electron microscopy structure of chimeric influenza cH5/1 hemagglutinin bound to head-targeting antibody, 3F5
Map dataTomographic subvolume average of virus-bound influenza cH5/1 HA in complex with the head-binding antibody, 3F5
Sample
  • Sample: Tomographic subvolume average of virus-bound influenza cH5/1 HA in complex with the head-binding antibody, 3F5
  • Protein or peptide: hemagglutinin
  • Protein or peptide: Immunoglobulin 3F5
KeywordsInfluenza / chimeric HA / universal vaccine candidate
Biological speciesunidentified influenza virus / Mus musculus (house mouse)
Methodsubtomogram averaging / cryo EM / Resolution: 25.0 Å
AuthorsTran EEH / Podolsky KA / Bartesaghi A / Kuybeda O / Grandinetti G / Wohlbold TJ / Tan GS / Nachbagauer R / Palese P / Krammer F / Subramaniam S
CitationJournal: mBio / Year: 2016
Title: Cryo-electron Microscopy Structures of Chimeric Hemagglutinin Displayed on a Universal Influenza Vaccine Candidate.
Authors: Erin E H Tran / Kira A Podolsky / Alberto Bartesaghi / Oleg Kuybeda / Giovanna Grandinetti / Teddy John Wohlbold / Gene S Tan / Raffael Nachbagauer / Peter Palese / Florian Krammer / Sriram Subramaniam /
Abstract: Influenza viruses expressing chimeric hemagglutinins (HAs) are important tools in the quest for a universal vaccine. Using cryo-electron tomography, we have determined the structures of a chimeric HA ...Influenza viruses expressing chimeric hemagglutinins (HAs) are important tools in the quest for a universal vaccine. Using cryo-electron tomography, we have determined the structures of a chimeric HA variant that comprises an H1 stalk and an H5 globular head domain (cH5/1 HA) in native and antibody-bound states. We show that cH5/1 HA is structurally different from native HA, displaying a 60° rotation between the stalk and head groups, leading to a novel and unexpected "open" arrangement of HA trimers. cH5/1N1 viruses also display higher glycoprotein density than pH1N1 or H5N1 viruses, but despite these differences, antibodies that target either the stalk or head domains of hemagglutinins still bind to cH5/1 HA with the same consequences as those observed with native H1 or H5 HA. Our results show that a large range of structural plasticity can be tolerated in the chimeric spike scaffold without disrupting structural and geometric aspects of antibody binding.
IMPORTANCE: Chimeric hemagglutinin proteins are set to undergo human clinical trials as a universal influenza vaccine candidate, yet no structural information for these proteins is available. Using ...IMPORTANCE: Chimeric hemagglutinin proteins are set to undergo human clinical trials as a universal influenza vaccine candidate, yet no structural information for these proteins is available. Using cryo-electron tomography, we report the first three-dimensional (3D) visualization of chimeric hemagglutinin proteins displayed on the surface of the influenza virus. We show that, unexpectedly, the chimeric hemagglutinin structure differs from those of naturally occurring hemagglutinins by displaying a more open head domain and a dramatically twisted head/stalk arrangement. Despite this unusual spatial relationship between head and stalk regions, virus preparations expressing the chimeric hemagglutinin are fully infectious and display a high glycoprotein density, which likely helps induction of a broadly protective immune response.
History
DepositionFeb 29, 2016-
Header (metadata) releaseMar 30, 2016-
Map releaseMar 30, 2016-
UpdateApr 13, 2016-
Current statusApr 13, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0086
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0086
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_6613.map.gz / Format: CCP4 / Size: 908.2 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTomographic subvolume average of virus-bound influenza cH5/1 HA in complex with the head-binding antibody, 3F5
Voxel sizeX=Y=Z: 4.1 Å
Density
Contour LevelBy AUTHOR: 0.0086 / Movie #1: 0.0086
Minimum - Maximum-0.01649853 - 0.02955358
Average (Standard dev.)-0.00005018 (±0.00371463)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions494999
Spacing494999
CellA: 200.9 Å / B: 200.9 Å / C: 405.9 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.14.14.1
M x/y/z494999
origin x/y/z0.0000.0000.000
length x/y/z200.900200.900405.900
α/β/γ90.00090.00090.000
start NX/NY/NZ-8211244
NX/NY/NZ115138149
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS494999
D min/max/mean-0.0160.030-0.000

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Supplemental data

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Sample components

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Entire : Tomographic subvolume average of virus-bound influenza cH5/1 HA i...

EntireName: Tomographic subvolume average of virus-bound influenza cH5/1 HA in complex with the head-binding antibody, 3F5
Components
  • Sample: Tomographic subvolume average of virus-bound influenza cH5/1 HA in complex with the head-binding antibody, 3F5
  • Protein or peptide: hemagglutinin
  • Protein or peptide: Immunoglobulin 3F5

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Supramolecule #1000: Tomographic subvolume average of virus-bound influenza cH5/1 HA i...

SupramoleculeName: Tomographic subvolume average of virus-bound influenza cH5/1 HA in complex with the head-binding antibody, 3F5
type: sample / ID: 1000 / Number unique components: 2

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Macromolecule #1: hemagglutinin

MacromoleculeName: hemagglutinin / type: protein_or_peptide / ID: 1 / Oligomeric state: trimer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: unidentified influenza virus / Strain: cH5/1N1

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Macromolecule #2: Immunoglobulin 3F5

MacromoleculeName: Immunoglobulin 3F5 / type: protein_or_peptide / ID: 2 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Mus musculus (house mouse) / Strain: BALB/c / synonym: Mouse

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4 / Details: 25 mM Tris, 100 mM NaCl
GridDetails: Quantifoil Multi-A 200 mesh carbon grids
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 90 K / Instrument: LEICA EM GP
Timed resolved state: Virus was incubated with antibody for ~30 minutes on ice before solution was plunge-frozen.
Method: Blot for 6 seconds before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 23000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 64000
Specialist opticsEnergy filter - Name: Gatan, Inc. / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Sample stageSpecimen holder: Liquid nitrogen-cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 °
DateApr 21, 2015
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number real images: 16 / Average electron dose: 120 e/Å2
Details: Every image is the average of four frames recorded by the direct electron detector.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: OTHER / Software - Name: IMOD / Number subtomograms used: 99
DetailsSubtomograms were selected manually.

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