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- PDB-9tq7: Neuraminidase NA isolated from the H1N1 strain A/Victoria/2570/20... -

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Basic information

Entry
Database: PDB / ID: 9tq7
TitleNeuraminidase NA isolated from the H1N1 strain A/Victoria/2570/2019 propagated in eggs
ComponentsNeuraminidase
KeywordsHYDROLASE / glycosidase / influenza virus / viral release
Biological speciesInfluenza A virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.58 Å
AuthorsBorowska, A. / Kang, H. / Slotboom, D.J. / Daniels, R.
Funding support Netherlands, 1items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO) Netherlands
CitationJournal: J Biol Chem / Year: 2026
Title: INAAC: An affinity chromatography strategy enabling characterization and quantification of influenza neuraminidase antigens in vaccines.
Authors: Hyeog Kang / Anna M Borowska / Tapan Kanai / Jin Gao / Hai Yu / Xi Chen / Jason Gorman / Dirk-Jan Slotboom / Robert Daniels /
Abstract: Seasonal influenza vaccines are commonly produced using viruses containing hemagglutinin (HA) and neuraminidase (NA) antigens from recommended strains. However, NA amounts are not monitored due to ...Seasonal influenza vaccines are commonly produced using viruses containing hemagglutinin (HA) and neuraminidase (NA) antigens from recommended strains. However, NA amounts are not monitored due to lack of strategies for isolating NA reference antigens from viruses, limiting the ability to evaluate any contributions from NA for more than 50 years. Here, we developed an influenza neuraminidase active-site affinity chromatography (INAAC) strategy that uses an active-site binding antibody to isolate functional NAs from influenza A and B vaccine strains. INAAC recovered 20 to 60% of detergent-solubilized NA activity from vaccine viruses and recombinant sources, with CaCl elution proving most effective. ELISA results with the isolated NA from the H1N1 strain and recombinant full-length N1 showed that a commercial vaccine contains functional N1 and H1 at a ratio of ∼1:10. Structure determination of the isolated N1 by cryo-electron microscopy confirmed the native tetrameric conformation and provided insight into the stalk conformation of a virus-derived NA. Finally, comparative analyses of NAs isolated from recent egg-propagated vaccine strains (H1N1, H3N2, and type B) revealed a unique proteolytic susceptibility of type B NA and strain-specific differences in sialic acid affinities and catalytic rates. These results demonstrate that INAAC supports multiple applications from NA structural analysis to producing NA vaccine antigens and reference standards, addressing longstanding challenges for incorporating NA into influenza vaccine development and quality control.
History
DepositionDec 19, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 3, 2026Provider: repository / Type: Initial release
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Revision 1.1Jun 17, 2026Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Revision 1.1Jun 17, 2026Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / EM metadata / Category: citation / citation_author / em_admin / Data content type: EM metadata / EM metadata / EM metadata
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Neuraminidase
B: Neuraminidase
C: Neuraminidase
D: Neuraminidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)209,61924
Polymers206,6444
Non-polymers2,97520
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Neuraminidase


Mass: 51660.902 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Influenza A virus (A/(H1N1)) / References: exo-alpha-sialidase
#2: 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.: 12 / Source method: obtained synthetically / Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
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
#3: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
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: Neuraminidase NA isolated from the H1N1 strain A/Victoria/2570/2019 propagated in eggs
Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.2142 MDa / Experimental value: NO
Source (natural)Organism: Influenza A virus (A/(H1N1))
Buffer solutionpH: 7
Details: 20 mM HEPES pH 7, 150 mM NaCl, 1 mM CaCl2, 0.0001% HDM
SpecimenConc.: 0.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 5 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 288 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Calibrated magnification: 48924 X / Nominal defocus max: 1900 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 57.3 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2282
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 60 / Used frames/image: 1-60

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Processing

EM software
IDNameVersionCategory
1crYOLO1.7.6particle selection
2EPU3.2image acquisition
4CTFFIND4.1.14CTF correction
7Coot0.9model fitting
9cryoSPARC4.5.1initial Euler assignment
10RELION5final Euler assignment
12RELION53D reconstruction
13PHENIX1.21.2_5419model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 456795
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 3.58 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 204391 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 3NSS

3nss


Pdb chain-ID: A / Accession code: 3NSS / Source name: PDB / Type: experimental model
RefinementHighest resolution: 3.58 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00312480
ELECTRON MICROSCOPYf_angle_d0.62416936
ELECTRON MICROSCOPYf_dihedral_angle_d6.0531931
ELECTRON MICROSCOPYf_chiral_restr0.051796
ELECTRON MICROSCOPYf_plane_restr0.0032184

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