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- PDB-9fm2: Cryo-EM structure of Influenza B/Washington/02/2019 virus neurami... -

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Basic information

Entry
Database: PDB / ID: 9fm2
TitleCryo-EM structure of Influenza B/Washington/02/2019 virus neuraminidase in complex with single-domain antibody hVHH-525.
Components
  • Neuraminidase
  • Single-domain antibody hVHH-525
KeywordsANTIVIRAL PROTEIN / neuraminidase / single-domain antibody / complex
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 speciesLama glama (llama)
Influenza B virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsFelix, J. / Matthys, A. / Savvides, S.N. / Saelens, X.
Funding support Belgium, 3items
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, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 9, 2025Provider: repository / Type: Initial release
Revision 1.1Jul 16, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.page_last / _citation.pdbx_database_id_PubMed ..._citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Single-domain antibody hVHH-525
B: Neuraminidase
C: Neuraminidase
D: Neuraminidase
E: Neuraminidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)228,1439
Polymers227,2585
Non-polymers8854
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable, light scattering, SEC-MALS
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area11330 Å2
ΔGint-50 kcal/mol
Surface area54160 Å2

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Components

#1: Antibody Single-domain antibody hVHH-525


Mass: 13094.380 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Komagataella phaffii (fungus)
#2: Protein
Neuraminidase


Mass: 53540.992 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza B virus / Gene: NA / Cell line (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: A0A5J6DRQ4, exo-alpha-sialidase
#3: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Complex between Influenza B/Washington/02/2019 virus neuraminidase (tetramer) and one copy of single-domain antibody hVHH-525
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.264 MDa / Experimental value: YES
Source (natural)Organism: Influenza B virus
Source (recombinant)Organism: Homo sapiens (human) / Cell: Expi293F
Buffer solutionpH: 7.4
Buffer component
IDConc.NameBuffer-ID
10.137 MSodium chloride1
20.0027 MPotassium Chloride1
30.01 MSodium Phosphate Dibasic1
40.0018 MPotassium Phosphate Monobasic1
SpecimenConc.: 0.12 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 28057

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Processing

EM software
IDNameVersionCategory
1crYOLOparticle selection
2SerialEMimage acquisition
4cryoSPARC4.2CTF correction
10cryoSPARC4.2initial Euler assignment
11cryoSPARC4.2final Euler assignment
12cryoSPARC4.2classification
13cryoSPARC4.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 697697
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 158674
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.
Num. of class averages: 1 / Symmetry type: POINT
RefinementResolution: 3.6→123.84 Å / Cor.coef. Fo:Fc: 0.932 / SU B: 34.585 / SU ML: 0.484 / ESU R: 0.868
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.34089 --
obs0.34089 69384 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 184.806 Å2
Refinement stepCycle: 1 / Total: 11567
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0090.01211866
ELECTRON MICROSCOPYr_bond_other_d00.01610830
ELECTRON MICROSCOPYr_angle_refined_deg1.3521.82416053
ELECTRON MICROSCOPYr_angle_other_deg0.4761.76525029
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.79251473
ELECTRON MICROSCOPYr_dihedral_angle_2_deg2.717579
ELECTRON MICROSCOPYr_dihedral_angle_3_deg14.57101956
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.0680.21726
ELECTRON MICROSCOPYr_gen_planes_refined0.0060.0213958
ELECTRON MICROSCOPYr_gen_planes_other0.0010.022730
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it29.2817.6955949
ELECTRON MICROSCOPYr_mcbond_other29.26717.6965949
ELECTRON MICROSCOPYr_mcangle_it45.6931.6457403
ELECTRON MICROSCOPYr_mcangle_other45.68831.6447404
ELECTRON MICROSCOPYr_scbond_it30.00120.0115917
ELECTRON MICROSCOPYr_scbond_other29.99820.0125918
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other46.36835.7658651
ELECTRON MICROSCOPYr_long_range_B_refined62.647215.2444296
ELECTRON MICROSCOPYr_long_range_B_other62.646215.2444297
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.6→3.693 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork1.532 5113 -
obs--100 %

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