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- EMDB-7021: Structure of influenza hemagglutinin from A/New Caledonia/20/99 i... -

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Basic information

Entry
Database: EMDB / ID: EMD-7021
TitleStructure of influenza hemagglutinin from A/New Caledonia/20/99 in complex with FAB 441D6.
Map dataHemagglutinin ectodomain derived from influenza A/New Caledonia/20/99 expressed with a trimerization domain (foldon), in complex with 3 molecules of FAB 441D6.
Sample
  • Complex: Influenza hemagglutinin from A/New Caledonia/20/99, in complex with 3 molecules of FAB 441D6.
    • Protein or peptide: Influenza hemagglutinin from A/New Caledonia/20/99, with C-terminal trimerization domain (T4 fibritin foldon).
Biological speciesInfluenza A virus
Methodsingle particle reconstruction / cryo EM / Resolution: 8.0 Å
AuthorsGallagher JR / Harris AK
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases1ZIAAI001180 United States
CitationJournal: Nat Immunol / Year: 2019
Title: Mosaic nanoparticle display of diverse influenza virus hemagglutinins elicits broad B cell responses.
Authors: Masaru Kanekiyo / M Gordon Joyce / Rebecca A Gillespie / John R Gallagher / Sarah F Andrews / Hadi M Yassine / Adam K Wheatley / Brian E Fisher / David R Ambrozak / Adrian Creanga / Kwanyee ...Authors: Masaru Kanekiyo / M Gordon Joyce / Rebecca A Gillespie / John R Gallagher / Sarah F Andrews / Hadi M Yassine / Adam K Wheatley / Brian E Fisher / David R Ambrozak / Adrian Creanga / Kwanyee Leung / Eun Sung Yang / Seyhan Boyoglu-Barnum / Ivelin S Georgiev / Yaroslav Tsybovsky / Madhu S Prabhakaran / Hanne Andersen / Wing-Pui Kong / Ulrich Baxa / Kathryn L Zephir / Julie E Ledgerwood / Richard A Koup / Peter D Kwong / Audray K Harris / Adrian B McDermott / John R Mascola / Barney S Graham /
Abstract: The present vaccine against influenza virus has the inevitable risk of antigenic discordance between the vaccine and the circulating strains, which diminishes vaccine efficacy. This necessitates new ...The present vaccine against influenza virus has the inevitable risk of antigenic discordance between the vaccine and the circulating strains, which diminishes vaccine efficacy. This necessitates new approaches that provide broader protection against influenza. Here we designed a vaccine using the hypervariable receptor-binding domain (RBD) of viral hemagglutinin displayed on a nanoparticle (np) able to elicit antibody responses that neutralize H1N1 influenza viruses spanning over 90 years. Co-display of RBDs from multiple strains across time, so that the adjacent RBDs are heterotypic, provides an avidity advantage to cross-reactive B cells. Immunization with the mosaic RBD-np elicited broader antibody responses than those induced by an admixture of nanoparticles encompassing the same set of RBDs as separate homotypic arrays. Furthermore, we identified a broadly neutralizing monoclonal antibody in a mouse immunized with mosaic RBD-np. The mosaic antigen array signifies a unique approach that subverts monotypic immunodominance and allows otherwise subdominant cross-reactive B cell responses to emerge.
History
DepositionSep 8, 2017-
Header (metadata) releaseNov 1, 2017-
Map releaseNov 21, 2018-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0452
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0452
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7021.png

Downloads & links

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Map

FileDownload / File: emd_7021.map.gz / Format: CCP4 / Size: 67 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHemagglutinin ectodomain derived from influenza A/New Caledonia/20/99 expressed with a trimerization domain (foldon), in complex with 3 molecules of FAB 441D6.
Voxel sizeX=Y=Z: 1.3797 Å
Density
Contour LevelBy AUTHOR: 0.0452 / Movie #1: 0.0452
Minimum - Maximum-0.05205744 - 0.20475802
Average (Standard dev.)-0.00022363848 (±0.009204472)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions260260260
Spacing260260260
CellA=B=C: 358.722 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.37971.37971.3797
M x/y/z260260260
origin x/y/z0.0000.0000.000
length x/y/z358.722358.722358.722
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ320320320
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS260260260
D min/max/mean-0.0520.205-0.000

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Supplemental data

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Mask #1

Fileemd_7021_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: RELION unfiltered half map (1)

Fileemd_7021_half_map_1.map
AnnotationRELION unfiltered half map (1)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: RELION unfiltered half map (2)

Fileemd_7021_half_map_2.map
AnnotationRELION unfiltered half map (2)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Influenza hemagglutinin from A/New Caledonia/20/99, in complex wi...

EntireName: Influenza hemagglutinin from A/New Caledonia/20/99, in complex with 3 molecules of FAB 441D6.
Components
  • Complex: Influenza hemagglutinin from A/New Caledonia/20/99, in complex with 3 molecules of FAB 441D6.
    • Protein or peptide: Influenza hemagglutinin from A/New Caledonia/20/99, with C-terminal trimerization domain (T4 fibritin foldon).

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Supramolecule #1: Influenza hemagglutinin from A/New Caledonia/20/99, in complex wi...

SupramoleculeName: Influenza hemagglutinin from A/New Caledonia/20/99, in complex with 3 molecules of FAB 441D6.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Influenza A virus / Strain: A/New Caledonia/20/99
Recombinant expressionOrganism: Homo sapiens (human)

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Macromolecule #1: Influenza hemagglutinin from A/New Caledonia/20/99, with C-termin...

MacromoleculeName: Influenza hemagglutinin from A/New Caledonia/20/99, with C-terminal trimerization domain (T4 fibritin foldon).
type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Influenza A virus / Strain: A/New Caledonia/20/99
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DTICIGYHAN NSTDTVDTVL EKNVTVTHSV NLLEDSHNGK LCRLKGTAPL QLGNCSVAGW ILGNPECESL FSKESWSYIA ETPNPENGTC YPGYFADYEE LREQLSSVSS FERFEIFPKE SSWPNHTVTK GVTASCSHNG KSSFYKNLLW LTEKNGLYPN LSKSYVNNKE ...String:
DTICIGYHAN NSTDTVDTVL EKNVTVTHSV NLLEDSHNGK LCRLKGTAPL QLGNCSVAGW ILGNPECESL FSKESWSYIA ETPNPENGTC YPGYFADYEE LREQLSSVSS FERFEIFPKE SSWPNHTVTK GVTASCSHNG KSSFYKNLLW LTEKNGLYPN LSKSYVNNKE KEVLVLWGVH HPSNIGDQRA IYHTENAYVS VVSSHYSRRF TPEITKRPKV RDQEGRINYY WTLLEPGDTI IFEANGNLIA PWYAFALSRG FGSGIITSNA SMGECDAKCQ TPQGAINSSL PFQNVHPVTI GECPKYVRST KLRMVTGLRN IPSIQSRGLF GAIAGFIEGG WTGMIDGWYG YHHQNEQGSG YAADQKSTQN AIDGITNKVN SVIEKMNTQF TAVGKEFNKL ERRMENLNKK VDDGFLDIWT YNAELLVLLE NERTLDFHDS NVKNLYEKVK TQLKNNAKEI GNGCFEFYHK CNNECMESVK NGTYDYPKYS EESKLNREKI DGSGLVPRGS GYIPEAPRDG QAYVRKDGEW VLLSTFLGSA HIVMVDAYKP TKGHHHHHH

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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

Concentration1 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
1.8 mMKH2PO4potassium phosphate
10.0 mMNa2HPO4sodium phosphate
137.0 mMNaClsodium chloride
2.7 mMKClpotassium chloride

Details: PBS
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 101.325 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
DetailsComplexes of hemagglutinin and FAB were purified from unbound components by gel filtration.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Frames/image: 1-7 / Number grids imaged: 1 / Number real images: 464 / Average exposure time: 1.0 sec. / Average electron dose: 34.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 8.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 202399
Details: RELION autopicking was performed using templates from RELION 2D class averages of manually picked particles.
CTF correctionSoftware - Name: CTFFIND4 (ver. 4.1.5)
Details: CTF values determined with CTFFIND4, and CTF correction was applied by RELION.
Startup modelType of model: OTHER
Details: EMAN2 e2initialmodel.py was used to generate an initial model from RELION 2D class averages.
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 8.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION2 (ver. 3/20/2017)
Details: The final map was re-refined with RELION2 released 8/18/2017, after a bug was patched affecting fidelity of the "gold-standard" refinement procedure. No changes were found in the resulting ...Details: The final map was re-refined with RELION2 released 8/18/2017, after a bug was patched affecting fidelity of the "gold-standard" refinement procedure. No changes were found in the resulting map, which refined to an identical resolution.
Number images used: 75233
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: RELION2 (ver. 3/20/2017)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION2 (ver. 3/20/2017) / Software - details: relion_refine
FSC plot (resolution estimation)

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