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- EMDB-50547: Cryo-EM structure of Influenza B/Washington/02/2019 virus neurami... -

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Basic information

Entry
Database: EMDB / ID: EMD-50547
TitleCryo-EM structure of Influenza B/Washington/02/2019 virus neuraminidase in complex with single-domain antibody hVHH-525.
Map dataSharpened map of Influenza B/Washington/02/2019 virus Neuraminidase in complex with single-chain antibody hVHH-525.
Sample
  • Complex: Complex between Influenza B/Washington/02/2019 virus neuraminidase (tetramer) and one copy of single-domain antibody hVHH-525
    • Protein or peptide: Single-domain antibody hVHH-525
    • Protein or peptide: Neuraminidase
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Keywordsneuraminidase / single-domain antibody / complex / ANTIVIRAL PROTEIN
Function / homology
Function and homology information


exo-alpha-sialidase / exo-alpha-sialidase activity / carbohydrate metabolic process / host cell plasma membrane / virion membrane / metal ion binding / membrane
Similarity search - Function
Glycoside hydrolase, family 34 / Neuraminidase / Sialidase superfamily
Similarity search - Domain/homology
Biological speciesInfluenza B virus / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsFelix J / Matthys A / Savvides SN / Saelens X
Funding support Belgium, 3 items
OrganizationGrant numberCountry
Research Foundation - Flanders (FWO)1S93223N Belgium
Other privateFlanders Institute for Biotechnology (VIB) grant C0101 Belgium
Other privateFlanders Institute for Biotechnology (VIB) grant C0301 Belgium
CitationJournal: Nat Commun / Year: 2025
Title: Single-domain antibodies directed against hemagglutinin and neuraminidase protect against influenza B viruses.
Authors: Arne Matthys / Jan Felix / Joao Paulo Portela Catani / Kenny Roose / Wim Nerinckx / Benthe Van Buyten / Daria Fijalkowska / Nico Callewaert / Savvas N Savvides / Xavier Saelens /
Abstract: Influenza B viruses are antigenically diverse and contribute significantly to the annual influenza burden. Here we report influenza B virus neutralizing single-domain antibodies that target highly ...Influenza B viruses are antigenically diverse and contribute significantly to the annual influenza burden. Here we report influenza B virus neutralizing single-domain antibodies that target highly conserved regions of the hemagglutinin and neuraminidase. Structural studies by single particle electron cryo-microscopy (cryo-EM) revealed that one of these single-domain antibodies prevents the conformational transition of the viral hemagglutinin to the post-fusion state by targeting a quaternary epitope spanning two protomers in the hemagglutinin-stem region. A second single-domain antibody broadly inhibits influenza B neuraminidase activity, including an oseltamivir-resistant neuraminidase, and its complex with neuraminidase elucidated by single particle cryo-EM established that it binds to residues in the neuraminidase catalytic site. Head-to-tail fusions of these single-domain antibodies led to bispecific binders that further improved the neutralization breadth and potency against influenza B viruses. These single-domain antibodies, fused to a human IgG1-Fc domain, fully protected female mice against an otherwise lethal influenza B virus challenge. Our findings underscore the potential of engineered single-domain antibodies to help control influenza B virus infections.
History
DepositionJun 5, 2024-
Header (metadata) releaseJul 9, 2025-
Map releaseJul 9, 2025-
UpdateJul 16, 2025-
Current statusJul 16, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50547.png

Downloads & links

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Map

FileDownload / File: emd_50547.map.gz / Format: CCP4 / Size: 10.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map of Influenza B/Washington/02/2019 virus Neuraminidase in complex with single-chain antibody hVHH-525.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.44 Å/pix.
x 140 pix.
= 201.6 Å		1.44 Å/pix.
x 140 pix.
= 201.6 Å		1.44 Å/pix.
x 140 pix.
= 201.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.44 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.14
Minimum - Maximum-0.6656399 - 0.9984864
Average (Standard dev.)0.0031092467 (±0.0380508)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions140140140
Spacing140140140
CellA=B=C: 201.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_50547_msk_1.map
Projections & Slices
AxesZYX

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Additional map: Unsharpened, unfiltered full map of Influenza B/Washington/02/2019 virus...

Fileemd_50547_additional_1.map
AnnotationUnsharpened, unfiltered full map of Influenza B/Washington/02/2019 virus Neuraminidase in complex with single-chain antibody hVHH-525, used for map and model validation.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: DeepEMhancer sharpened map of Influenza B/Washington/02/2019 virus Neuraminidase...

Fileemd_50547_additional_2.map
AnnotationDeepEMhancer sharpened map of Influenza B/Washington/02/2019 virus Neuraminidase in complex with single-chain antibody hVHH-525, used for model building and map visualization.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Local sharpened map of Influenza B/Washington/02/2019 virus Neuraminidase...

Fileemd_50547_additional_3.map
AnnotationLocal sharpened map of Influenza B/Washington/02/2019 virus Neuraminidase in complex with single-chain antibody hVHH-525, used for model refinement in Phenix.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 of Influenza B/Washington/02/2019 virus Neuraminidase...

Fileemd_50547_half_map_1.map
AnnotationHalf map 2 of Influenza B/Washington/02/2019 virus Neuraminidase in complex with single-chain antibody hVHH-525.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 of Influenza B/Washington/02/2019 virus Neuraminidase...

Fileemd_50547_half_map_2.map
AnnotationHalf map 1 of Influenza B/Washington/02/2019 virus Neuraminidase in complex with single-chain antibody hVHH-525.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex between Influenza B/Washington/02/2019 virus neuraminidas...

EntireName: Complex between Influenza B/Washington/02/2019 virus neuraminidase (tetramer) and one copy of single-domain antibody hVHH-525
Components
  • Complex: Complex between Influenza B/Washington/02/2019 virus neuraminidase (tetramer) and one copy of single-domain antibody hVHH-525
    • Protein or peptide: Single-domain antibody hVHH-525
    • Protein or peptide: Neuraminidase
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Complex between Influenza B/Washington/02/2019 virus neuraminidas...

SupramoleculeName: Complex between Influenza B/Washington/02/2019 virus neuraminidase (tetramer) and one copy of single-domain antibody hVHH-525
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Influenza B virus
Molecular weightTheoretical: 264 KDa

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Macromolecule #1: Single-domain antibody hVHH-525

MacromoleculeName: Single-domain antibody hVHH-525 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 13.09438 KDa
Recombinant expressionOrganism: Komagataella phaffii (fungus)
SequenceString:
DVQLVESGGG LVQPGGSLRL SCAASGDIFS THIMGWYRQA PGKQRELVAT IDTDGSTNYA DSVKGRFTIS RDNAKNTVYL QMNSLRPED TAVYYCNAES AYSDYRLPDY WGQGTLVTVS S

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Macromolecule #2: Neuraminidase

MacromoleculeName: Neuraminidase / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO / EC number: exo-alpha-sialidase
Source (natural)Organism: Influenza B virus
Molecular weightTheoretical: 53.540992 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MPMGSLQPLA TLYLLGMLVA SVLSAWSHPQ FEKGSGIINE TADDIVYRLT VIIDDRYESL KNLITLRADR LEMIINDNVS TILASIGSG VTLLLPEPEW TYPRLSCPGS TFQKALLISP HRFGETKGNS APLIIREPFV ACGPNECKHF ALTHYAAQPG G YYNGTRGD ...String:
MPMGSLQPLA TLYLLGMLVA SVLSAWSHPQ FEKGSGIINE TADDIVYRLT VIIDDRYESL KNLITLRADR LEMIINDNVS TILASIGSG VTLLLPEPEW TYPRLSCPGS TFQKALLISP HRFGETKGNS APLIIREPFV ACGPNECKHF ALTHYAAQPG G YYNGTRGD RNKLRHLISV KLGKIPTVEN SIFHMAAWSG SACHDGKEWT YIGVDGPDNN ALLKVKYGEA YTDTYHSYAN NI LRTQESA CNCIGGNCYL MITDGSASGV SECRFLKIRE GRIIKEIFPT GRVKHTEECT CGFASNKTIE CACRDNRYTA KRP FVKLNV ETDTAEIRLM CTDTYLDTPR PNDGSITGPC ESDGDKGSGG IKGGFVHQRM KSKIGRWYSR TMSKTERMGM GLYV KYGGD PWADSDALTF SGVMVSMKEP GWYSFGFEIK DKKCDVPCIG IEMVHDGGKE TWHSAATAIY CLMGSGQLLW DTVTG VDMA L

UniProtKB: Neuraminidase

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Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 4 / 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

Concentration0.12 mg/mL
BufferpH: 7.4
Component:
ConcentrationName
0.137 MSodium chloride
0.0027 MPotassium Chloride
0.01 MSodium Phosphate Dibasic
0.0018 MPotassium Phosphate Monobasic
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 28057 / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm

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Image processing

Particle selectionNumber selected: 697697
CTF correctionSoftware - Name: cryoSPARC (ver. 4.2) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2)
Details: Compositional heterogeneity of the sample necessitated the use of a 3D processing strategy combining symmetry expansion, focused 3D classification without alignment, signal subtraction and ...Details: Compositional heterogeneity of the sample necessitated the use of a 3D processing strategy combining symmetry expansion, focused 3D classification without alignment, signal subtraction and localized refinement. The final particle set consists of 158674 symmetry expanded and signal subtracted particles.
Number images used: 158674
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2)
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC (ver. 4.2)
FSC plot (resolution estimation)

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