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- EMDB-45637: CryoEM structure of NC99 hemagglutinin trimer in complex with Fab... -

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Basic information

Entry
Database: EMDB / ID: EMD-45637
TitleCryoEM structure of NC99 hemagglutinin trimer in complex with Fab T009 3-E04
Map data
Sample
  • Complex: NC99 HA in complex with Fab T009 3-E04
    • Protein or peptide: Hemagglutinin HA1 chain
    • Protein or peptide: Hemagglutinin HA2 chain
    • Protein or peptide: 3-E04 Heavy chain
    • Protein or peptide: 3-E04 Light chain
KeywordsImmune system / Hemagglutinin / Fab / Macaque / VIRAL PROTEIN
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 / Macaca fascicularis (crab-eating macaque)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsLi N / Tsybovsky Y / Sangesland M / Kanekiyo M
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID) United States
CitationJournal: bioRxiv / Year: 2025
Title: Functional, Immunogenetic, and Structural Convergence in Influenza Immunity between Humans and Macaques.
Authors: Maya Sangesland / Ning Li / Yaroslav Tsybovsky / Megan D Rodgers / Julianna Han / Alesandra J Rodriguez / James A Ferguson / Amy R Henry / Sarah C Smith / Jesmine Roberts-Torres / Rebecca A ...Authors: Maya Sangesland / Ning Li / Yaroslav Tsybovsky / Megan D Rodgers / Julianna Han / Alesandra J Rodriguez / James A Ferguson / Amy R Henry / Sarah C Smith / Jesmine Roberts-Torres / Rebecca A Gillespie / Cuiping Liu / Jonah S Merriam / Tyler Stephens / Connor Williams / Emma Maestle / Martin Corcoran / Michelle Ravichandran / Adrian Creanga / Sarah F Andrews / Theodore C Pierson / Gunilla B Karlsson Hedestam / Chaim A Schramm / Douglas S Reed / Daniel C Douek / Tongqing Zhou / Andrew B Ward / Masaru Kanekiyo
Abstract: Human B cell immunity to the influenza hemagglutinin (HA) stem region, a universal influenza vaccine target, is often stereotyped and immunogenetically restricted, posing challenges for study outside ...Human B cell immunity to the influenza hemagglutinin (HA) stem region, a universal influenza vaccine target, is often stereotyped and immunogenetically restricted, posing challenges for study outside humans. Here, we show that macaques vaccinated with a HA stem immunogen elicit human-like public B cell lineages targeting two major conserved sites of vulnerability, the central stem and anchor epitopes. Central stem antibodies were predominantly derived from V 1-138, the macaque homolog of human V 1-69, a V -gene preferentially used in human central stem broadly neutralizing antibodies (bnAbs). Similarly, macaques produced anchor bnAbs with the human-like NWP motif. Both bnAb lineages were functionally and structurally analogous to their human counterparts, with recognition mediated largely by germline-encoded motifs. Thus the macaque immunoglobulin repertoire supports human-like public bnAb responses to influenza HA. Moreover, this underscores the utility of homologous germline-encoded immunity, suggesting that immune repertoires of macaques and humans may have been similarly shaped during evolution.
HIGHLIGHTS: Functional human-like public antibody lineages can be elicited to HA stem supersites in macaques. Macaque central stem bnAbs are predominantly derived from V 1-138, a V -gene homologous ...HIGHLIGHTS: Functional human-like public antibody lineages can be elicited to HA stem supersites in macaques. Macaque central stem bnAbs are predominantly derived from V 1-138, a V -gene homologous to human V 1-69. The human-like CDR L3 NWP anchor epitope-targeting lineage can be elicited in macaques.Central stem and anchor bnAbs from humans and macaques engage their respective epitopes with atomic level similarity.
History
DepositionJul 8, 2024-
Header (metadata) releaseJul 9, 2025-
Map releaseJul 9, 2025-
UpdateJul 9, 2025-
Current statusJul 9, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45637.png

Downloads & links

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Map

FileDownload / File: emd_45637.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.11 Å/pix.
x 360 pix.
= 399.6 Å		1.11 Å/pix.
x 360 pix.
= 399.6 Å		1.11 Å/pix.
x 360 pix.
= 399.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.11 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-1.3807845 - 1.7718043
Average (Standard dev.)0.0003542048 (±0.02489391)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 399.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_45637_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_45637_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_45637_half_map_2.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 : NC99 HA in complex with Fab T009 3-E04

EntireName: NC99 HA in complex with Fab T009 3-E04
Components
  • Complex: NC99 HA in complex with Fab T009 3-E04
    • Protein or peptide: Hemagglutinin HA1 chain
    • Protein or peptide: Hemagglutinin HA2 chain
    • Protein or peptide: 3-E04 Heavy chain
    • Protein or peptide: 3-E04 Light chain

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Supramolecule #1: NC99 HA in complex with Fab T009 3-E04

SupramoleculeName: NC99 HA in complex with Fab T009 3-E04 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Influenza A virus
Molecular weightTheoretical: 330 KDa

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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
Molecular weightTheoretical: 36.417867 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 PSIQSR

UniProtKB: Hemagglutinin

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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
Molecular weightTheoretical: 25.28809 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GLFGAIAGFI EGGWTGMVDG WYGYHHQNEQ GSGYAADQKS TQNAINQITN KVNSVIEKMN TQFTAVGKEF NKLERRMENL NKKVDDGFL DIWTYNAELL VLLENERTLD FHDSNVKNLY EKVKSQLKNN AKEIGNGCFE FYHKCNNECM ESVKNGTYDY P KYSEESKL ...String:
GLFGAIAGFI EGGWTGMVDG WYGYHHQNEQ GSGYAADQKS TQNAINQITN KVNSVIEKMN TQFTAVGKEF NKLERRMENL NKKVDDGFL DIWTYNAELL VLLENERTLD FHDSNVKNLY EKVKSQLKNN AKEIGNGCFE FYHKCNNECM ESVKNGTYDY P KYSEESKL NREKIDGVSG RLVPRGSPGS GYIPEAPRDG QAYVRKDGEW VLLSTFLGHH HHHH

UniProtKB: Hemagglutinin

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Macromolecule #3: 3-E04 Heavy chain

MacromoleculeName: 3-E04 Heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Macaca fascicularis (crab-eating macaque)
Molecular weightTheoretical: 13.082574 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
EVQLVESGGG LVQPGGSLRL SCAASGFTFS SYGLSWVRQA PGKGLEWVSY ISNSGISTYY ADSVKGRFTI SRDNSKNTVS LQLNSLTPE DTAVYYCAKK GSTLIPYTWF DVWGPGVLVT VSS

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Macromolecule #4: 3-E04 Light chain

MacromoleculeName: 3-E04 Light chain / type: protein_or_peptide / ID: 4 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Macaca fascicularis (crab-eating macaque)
Molecular weightTheoretical: 11.560853 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
EIVMTQSPAT LSLSPGERAT LSCRASQSVS TSLAWYQQKP GQAPKLLIYG ASSRATGIPD RFSGSGSGTE FTLTISSLEP EDVGVYYCQ QDYNWPPLTF GGGTKVEIK

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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

Concentration0.01 mg/mL
BufferpH: 7.4
GridModel: Quantifoil R2/2 / Material: GOLD / Mesh: 400 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3840 pixel / Digitization - Dimensions - Height: 3712 pixel / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 22500
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: 2054500
CTF correctionSoftware - Name: CTFFIND (ver. 4.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.3.1) / Number images used: 160012
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8d21


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementProtocol: BACKBONE TRACE
Output model

PDB-9cjz:
CryoEM structure of NC99 hemagglutinin trimer in complex with Fab T009 3-E04

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