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- EMDB-6332: 3D reconstruction of a ferritin-based nanoparticle displaying H1 ... -

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Basic information

Entry
Database: EMDB / ID: EMD-6332
Title3D reconstruction of a ferritin-based nanoparticle displaying H1 Hemagglutinin stem epitopes
Map dataferritin-hemagglutinin nanoparticle
Sample
  • Sample: H. pylori ferritin fusion with engineered H1 hemagglutin stem domain
  • Protein or peptide: Hemagglutinin stem ferritin nanoparticle
Keywordsnanoparticle / vaccine / epitope display / influenza
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 16.0 Å
AuthorsGallagher JR / Harris AK / Yassine HM / Boyington JC / McTamney PM / Wei CJ / Masaru M / Kong WP / Wang L / Zhang Y ...Gallagher JR / Harris AK / Yassine HM / Boyington JC / McTamney PM / Wei CJ / Masaru M / Kong WP / Wang L / Zhang Y / Joyce MG / Lingwood D / Moin SM / Andersen H / Okuno Y / Rao SS / Kwong PD / Mascola JR / Nabel GJ / Graham BS
CitationJournal: Nat Med / Year: 2015
Title: Hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection.
Authors: Hadi M Yassine / Jeffrey C Boyington / Patrick M McTamney / Chih-Jen Wei / Masaru Kanekiyo / Wing-Pui Kong / John R Gallagher / Lingshu Wang / Yi Zhang / M Gordon Joyce / Daniel Lingwood / ...Authors: Hadi M Yassine / Jeffrey C Boyington / Patrick M McTamney / Chih-Jen Wei / Masaru Kanekiyo / Wing-Pui Kong / John R Gallagher / Lingshu Wang / Yi Zhang / M Gordon Joyce / Daniel Lingwood / Syed M Moin / Hanne Andersen / Yoshinobu Okuno / Srinivas S Rao / Audray K Harris / Peter D Kwong / John R Mascola / Gary J Nabel / Barney S Graham /
Abstract: The antibody response to influenza is primarily focused on the head region of the hemagglutinin (HA) glycoprotein, which in turn undergoes antigenic drift, thus necessitating annual updates of ...The antibody response to influenza is primarily focused on the head region of the hemagglutinin (HA) glycoprotein, which in turn undergoes antigenic drift, thus necessitating annual updates of influenza vaccines. In contrast, the immunogenically subdominant stem region of HA is highly conserved and recognized by antibodies capable of binding multiple HA subtypes. Here we report the structure-based development of an H1 HA stem-only immunogen that confers heterosubtypic protection in mice and ferrets. Six iterative cycles of structure-based design (Gen1-Gen6) yielded successive H1 HA stabilized-stem (HA-SS) immunogens that lack the immunodominant head domain. Antigenic characterization, determination of two HA-SS crystal structures in complex with stem-specific monoclonal antibodies and cryo-electron microscopy analysis of HA-SS on ferritin nanoparticles (H1-SS-np) confirmed the preservation of key structural elements. Vaccination of mice and ferrets with H1-SS-np elicited broadly cross-reactive antibodies that completely protected mice and partially protected ferrets against lethal heterosubtypic H5N1 influenza virus challenge despite the absence of detectable H5N1 neutralizing activity in vitro. Passive transfer of immunoglobulin from H1-SS-np-immunized mice to naive mice conferred protection against H5N1 challenge, indicating that vaccine-elicited HA stem-specific antibodies can protect against diverse group 1 influenza strains.
History
DepositionMay 4, 2015-
Header (metadata) releaseMay 27, 2015-
Map releaseAug 19, 2015-
UpdateSep 16, 2015-
Current statusSep 16, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.024
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.024
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6332.png

Downloads & links

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Map

FileDownload / File: emd_6332.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationferritin-hemagglutinin nanoparticle
Voxel sizeX=Y=Z: 2.63 Å
Density
Contour LevelBy AUTHOR: 0.024 / Movie #1: 0.024
Minimum - Maximum-0.04077554 - 0.1163357
Average (Standard dev.)0.00035891 (±0.01660633)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 336.64 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.632.632.63
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z336.640336.640336.640
α/β/γ90.00090.00090.000
start NX/NY/NZ-800-4
NX/NY/NZ1611358
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.0410.1160.000

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Supplemental data

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Segmentation: relion postprocess automask: threshold=0.008 extend=4, soft-edge=8,

Annotationrelion_postprocess automask: threshold=0.008 extend=4, soft-edge=8,
Fileemd_6332_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : H. pylori ferritin fusion with engineered H1 hemagglutin stem domain

EntireName: H. pylori ferritin fusion with engineered H1 hemagglutin stem domain
Components
  • Sample: H. pylori ferritin fusion with engineered H1 hemagglutin stem domain
  • Protein or peptide: Hemagglutinin stem ferritin nanoparticle

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Supramolecule #1000: H. pylori ferritin fusion with engineered H1 hemagglutin stem domain

SupramoleculeName: H. pylori ferritin fusion with engineered H1 hemagglutin stem domain
type: sample / ID: 1000 / Oligomeric state: nanoparticle of 24 subunits / Number unique components: 1
Molecular weightExperimental: 1 MDa / Theoretical: 970 KDa / Method: gel filtration

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Macromolecule #1: Hemagglutinin stem ferritin nanoparticle

MacromoleculeName: Hemagglutinin stem ferritin nanoparticle / type: protein_or_peptide / ID: 1 / Name.synonym: HA-stem ferritin nanoparticle / Number of copies: 24 / Oligomeric state: nanoparticle of 24 subunits / Recombinant expression: Yes
Source (natural)Organism: synthetic construct (others)
Molecular weightExperimental: 1 MDa / Theoretical: 970 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: 293F

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.8 mg/mL
BufferpH: 7.4 / Details: 12 mM Na/K phosphate, 137 mM NaCl
GridDetails: holey carbon film, Quantifoil
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 118 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 7.0 µm / Nominal defocus min: 3.5 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
TemperatureMin: 80 K / Max: 105 K / Average: 100 K
DateOct 1, 2014
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 209 / Average electron dose: 15 e/Å2 / Bits/pixel: 16
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: micrograph
Final two d classificationNumber classes: 80
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 16.0 Å / Resolution method: OTHER / Software - Name: CTFFIND3, RELION
Details: final reconstruction from 49% of the initial particle set
Number images used: 6540
DetailsParticles were selected manually.
FSC plot (resolution estimation)

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