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- EMDB-25040: Structure of H1 influenza hemagglutinin bound to Fab 310-39G10 -

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Basic information

Entry
Database: EMDB / ID: EMD-25040
TitleStructure of H1 influenza hemagglutinin bound to Fab 310-39G10
Map dataCryo-EM map of Fab 310-39G10 bound to H1 NC99 influenza hemagglutinin
Sample
  • Complex: Structure of H1 NC99 influenza hemagglutinin bound to Fab 310-39G10
    • Protein or peptide: Hemagglutinin HA1 chain
    • Protein or peptide: Hemagglutinin HA2 chain
    • Protein or peptide: 310-39G10 Fab, Heavy Chain
    • Protein or peptide: 310-39G10 Fab, Light Chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homology
Function and homology information


viral budding from plasma membrane / clathrin-dependent endocytosis of virus by host cell / host cell surface receptor binding / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / host cell plasma membrane / virion membrane / membrane
Similarity search - Function
Haemagglutinin, influenzavirus A / Haemagglutinin, HA1 chain, alpha/beta domain superfamily / Haemagglutinin / Haemagglutinin, influenzavirus A/B / Viral capsid/haemagglutinin protein
Similarity search - Domain/homology
Biological speciesInfluenza A virus / Influenza A virus (strain A/New Zealand:South Canterbury/35/2000 H1N1) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.37 Å
AuthorsTorrents de la Pena A / Ward AB
Funding support United States, 2 items
OrganizationGrant numberCountry
Bill & Melinda Gates FoundationOPP1170236 United States
Bill & Melinda Gates FoundationINV-004923 United States
CitationJournal: Immunity / Year: 2022
Title: Allelic polymorphism controls autoreactivity and vaccine elicitation of human broadly neutralizing antibodies against influenza virus.
Authors: Maya Sangesland / Alba Torrents de la Peña / Seyhan Boyoglu-Barnum / Larance Ronsard / Faez Amokrane Nait Mohamed / Thalia Bracamonte Moreno / Ralston M Barnes / Daniel Rohrer / Nils ...Authors: Maya Sangesland / Alba Torrents de la Peña / Seyhan Boyoglu-Barnum / Larance Ronsard / Faez Amokrane Nait Mohamed / Thalia Bracamonte Moreno / Ralston M Barnes / Daniel Rohrer / Nils Lonberg / Musie Ghebremichael / Masaru Kanekiyo / Andrew Ward / Daniel Lingwood /
Abstract: Human broadly neutralizing antibodies (bnAbs) targeting the hemagglutinin stalk of group 1 influenza A viruses (IAVs) are biased for IGHV1-69 alleles that use phenylalanine (F54) but not leucine (L54) ...Human broadly neutralizing antibodies (bnAbs) targeting the hemagglutinin stalk of group 1 influenza A viruses (IAVs) are biased for IGHV1-69 alleles that use phenylalanine (F54) but not leucine (L54) within their CDRH2 loops. Despite this, we demonstrated that both alleles encode for human IAV bnAbs that employ structurally convergent modes of contact to the same epitope. To resolve differences in lineage expandability, we compared F54 versus L54 as substrate within humanized mice, where antibodies develop with human-like CDRH3 diversity but are restricted to single V genes. While both alleles encoded for bnAb precursors, only F54 IGHV1-69 supported elicitation of heterosubtypic serum bnAbs following immunization with a stalk-only nanoparticle vaccine. L54 IGHV1-69 was unproductive, co-encoding for anergic B cells and autoreactive stalk antibodies that were cleared from B cell memory. Moreover, human stalk antibodies also demonstrated L54-dependent autoreactivity. Therefore, IGHV1-69 polymorphism, which is skewed ethnically, gates tolerance and vaccine expandability of influenza bnAbs.
History
DepositionSep 28, 2021-
Header (metadata) releaseAug 24, 2022-
Map releaseAug 24, 2022-
UpdateSep 28, 2022-
Current statusSep 28, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25040.png

Downloads & links

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Map

FileDownload / File: emd_25040.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of Fab 310-39G10 bound to H1 NC99 influenza hemagglutinin
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.15 Å/pix.
x 320 pix.
= 368. Å		1.15 Å/pix.
x 320 pix.
= 368. Å		1.15 Å/pix.
x 320 pix.
= 368. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.15 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0145
Minimum - Maximum-0.053279757 - 0.084932975
Average (Standard dev.)4.3829968e-07 (±0.0017858744)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 368.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Structure of H1 NC99 influenza hemagglutinin bound to Fab 310-39G10

EntireName: Structure of H1 NC99 influenza hemagglutinin bound to Fab 310-39G10
Components
  • Complex: Structure of H1 NC99 influenza hemagglutinin bound to Fab 310-39G10
    • Protein or peptide: Hemagglutinin HA1 chain
    • Protein or peptide: Hemagglutinin HA2 chain
    • Protein or peptide: 310-39G10 Fab, Heavy Chain
    • Protein or peptide: 310-39G10 Fab, Light Chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Structure of H1 NC99 influenza hemagglutinin bound to Fab 310-39G10

SupramoleculeName: Structure of H1 NC99 influenza hemagglutinin bound to Fab 310-39G10
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Influenza A virus
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: 293F

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Macromolecule #1: Hemagglutinin HA1 chain

MacromoleculeName: Hemagglutinin HA1 chain / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Influenza A virus (strain A/New Zealand:South Canterbury/35/2000 H1N1)
Strain: A/New Zealand:South Canterbury/35/2000 H1N1
Molecular weightTheoretical: 35.932309 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DTICIGYHAN NSTDTVDTVL EKNVTVTHSV NLLEDSHNGK LCLLKGIAPL QLGNCSVAGW ILGNPECELL ISKESWSYIV ETPNPENGT CYPGYFADYE ELREQLSSVS SFERFEIFPK ESSWPNHTVT GVSASCSHNG KSSFYRNLLW LTGKNGLYPN L SKSYVNNK ...String:
DTICIGYHAN NSTDTVDTVL EKNVTVTHSV NLLEDSHNGK LCLLKGIAPL QLGNCSVAGW ILGNPECELL ISKESWSYIV ETPNPENGT CYPGYFADYE ELREQLSSVS SFERFEIFPK ESSWPNHTVT GVSASCSHNG KSSFYRNLLW LTGKNGLYPN L SKSYVNNK EKEVLVLWGV HHPPNIGNQR ALYHTENAYV SVVSSHYSRR FTPEIAKRPK VRDQEGRINY YWTLLEPGDT II FEANGNL IAPWYAFALS RGFGSGIITS NAPMDECDAK CQTPQGAINS SLPFQNVHPV TIGECPKYVR SAKLRMVTGL RNI PS

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Macromolecule #2: Hemagglutinin HA2 chain

MacromoleculeName: Hemagglutinin HA2 chain / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Influenza A virus (strain A/New Zealand:South Canterbury/35/2000 H1N1)
Strain: A/New Zealand:South Canterbury/35/2000 H1N1
Molecular weightTheoretical: 26.637555 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: RETRGLFGAI AGFIEGGWTG MVDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLERR MENLNKKVD DGFLDIWTYN AELLVLLENE RTLDFHDSNV KNLYEKVKSQ LKNNAKEIGN GCFEFYHKCN NECMESVKNG T YDYPKYSE ...String:
RETRGLFGAI AGFIEGGWTG MVDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLERR MENLNKKVD DGFLDIWTYN AELLVLLENE RTLDFHDSNV KNLYEKVKSQ LKNNAKEIGN GCFEFYHKCN NECMESVKNG T YDYPKYSE ESKLNREKID GVKYIPEAPR DGQAYVRKDG EWVLLSTFLG SGLNDIFEAQ KIEWHEGHHH HHH

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Macromolecule #3: 310-39G10 Fab, Heavy Chain

MacromoleculeName: 310-39G10 Fab, Heavy Chain / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 12.796269 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
QVQLVQSGAE VKKPGSSVKV SCKASGGTFS RYTISWVRQA PGQGLEWMGR ISPIAGLENY AQKFQGRVTI TADISTSTAY MELSSLRSD DTAVYYCAGS SGYYQPPPYW GQGTLVTVSS

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Macromolecule #4: 310-39G10 Fab, Light Chain

MacromoleculeName: 310-39G10 Fab, Light Chain / type: protein_or_peptide / ID: 4 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.679963 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
EIVLTQAPFS LSLSPGERAT LSCRASQSVS SSSLAWYHQK PGQAPRLLIY GASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFSLYYC QQYGSSPLTF GQGTRLEIK

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Macromolecule #6: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 6 / Number of copies: 12 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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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

Concentration0.2 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
25.0 mMTris-HCl
150.0 mMNaClsodium chloride

Details: TBS buffer, PH 7.4
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 50.0 nm / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV
Details: Blotting time: 5.5 s Blotting force: 0 Waiting time: 7 s.
Detailshemagglutinin at 2mg/ml is complexed with the Fab at a molar ratio 1:3 (HA:Fab). The sample is incubated for 30min at RT and diluted with TBS to a final concentration of 0.2mg/ml

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 853 / Average exposure time: 10.0 sec. / Average electron dose: 49.88 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 36000
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4m4y

Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.37 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 135198
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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