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- EMDB-56127: Neuraminidase NA isolated from the H1N1 strain A/Victoria/2570/20... -

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Basic information

Entry
Database: EMDB / ID: EMD-56127
TitleNeuraminidase NA isolated from the H1N1 strain A/Victoria/2570/2019 propagated in eggs in complex with zanamivir
Map dataUnsharpened map of full-length vN1 in complex with zanamivir in HDM detergent
Sample
  • Complex: Neuraminidase NA isolated from the H1N1 strain A/Victoria/2570/2019 propagated in eggs in complex with zanamivir
    • Protein or peptide: Neuraminidase
  • Ligand: ZANAMIVIR
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CALCIUM ION
Keywordsglycosidase / influenza virus / viral release / HYDROLASE
Function / homology
Function and homology information


exo-alpha-sialidase / exo-alpha-sialidase activity / viral budding from plasma membrane / carbohydrate metabolic process / host cell plasma membrane / virion membrane / membrane / metal ion binding
Similarity search - Function
Sialidase, Influenza viruses A/B / Glycoside hydrolase, family 34 / Neuraminidase / Sialidase superfamily
Similarity search - Domain/homology
Biological speciesInfluenza A virus (A/(H1N1))
Methodsingle particle reconstruction / cryo EM / Resolution: 3.48 Å
AuthorsBorowska A / Kang H / Slotboom DJ / Daniels R
Funding support Netherlands, 1 items
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 Borowska / Tapan Kanai / Jin Gao / Hai Yu / Xi Chen / Jason Gorman / Dirk-Jan Slotboom / Robert Daniels /
Abstract: Seasonal influenza vaccines are produced using viruses containing the hemagglutinin (HA) and neuraminidase (NA) antigens. However, NA amounts have not been monitored for more than 50 years due to ...Seasonal influenza vaccines are produced using viruses containing the hemagglutinin (HA) and neuraminidase (NA) antigens. However, NA amounts have not been monitored for more than 50 years due to lack of strategies for isolating NA reference antigens from viruses, limiting the ability to evaluate any contributions from NA. 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 20-60% 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 by cryo-electron microscopy confirmed the native tetrameric conformation of the isolated N1 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, 2025-
Header (metadata) releaseJun 10, 2026-
Map releaseJun 10, 2026-
UpdateJun 10, 2026-
Current statusJun 10, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_56127.png

Downloads & links

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Map

FileDownload / File: emd_56127.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened map of full-length vN1 in complex with zanamivir in HDM detergent
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.02 Å/pix.
x 256 pix.
= 261.632 Å		1.02 Å/pix.
x 256 pix.
= 261.632 Å		1.02 Å/pix.
x 256 pix.
= 261.632 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.022 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00593
Minimum - Maximum-0.028150527 - 0.048175894
Average (Standard dev.)0.0000048675206 (±0.0011853656)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 261.632 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_56127_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: Unsharpened map of full-length vN1 in complex with...

Fileemd_56127_additional_1.map
AnnotationUnsharpened map of full-length vN1 in complex with zanamivir in HDM detergent (no symmetry applied)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 of full-length vN1 in complex...

Fileemd_56127_half_map_1.map
AnnotationHalf map 1 of full-length vN1 in complex with zanamivir in HDM detergent used for refinement and gold-standard FSC resolution calculation
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 of full-length vN1 in complex...

Fileemd_56127_half_map_2.map
AnnotationHalf map 2 of full-length vN1 in complex with zanamivir in HDM detergent used for refinement and gold-standard FSC resolution calculation
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Neuraminidase NA isolated from the H1N1 strain A/Victoria/2570/20...

EntireName: Neuraminidase NA isolated from the H1N1 strain A/Victoria/2570/2019 propagated in eggs in complex with zanamivir
Components
  • Complex: Neuraminidase NA isolated from the H1N1 strain A/Victoria/2570/2019 propagated in eggs in complex with zanamivir
    • Protein or peptide: Neuraminidase
  • Ligand: ZANAMIVIR
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CALCIUM ION

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Supramolecule #1: Neuraminidase NA isolated from the H1N1 strain A/Victoria/2570/20...

SupramoleculeName: Neuraminidase NA isolated from the H1N1 strain A/Victoria/2570/2019 propagated in eggs in complex with zanamivir
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Influenza A virus (A/(H1N1))
Molecular weightTheoretical: 214.2 KDa

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Macromolecule #1: Neuraminidase

MacromoleculeName: Neuraminidase / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: exo-alpha-sialidase
Source (natural)Organism: Influenza A virus (A/(H1N1))
Molecular weightTheoretical: 51.660902 KDa
SequenceString: MNPNQKIITI GSICMTIGTA NLILQIGNII SIWVSHSIQI GNQSQIETCN KSVITYENNT WVNQTFVNIS NTNSAARQSV ASVKLAGNS SLCPVSGWAI YSKDNSVRIG SKGDVFVIRE PFISCSPLEC RTFFLTQGAL LNDKHSNGTI KDRSPYRTLM S CPIGEVPS ...String:
MNPNQKIITI GSICMTIGTA NLILQIGNII SIWVSHSIQI GNQSQIETCN KSVITYENNT WVNQTFVNIS NTNSAARQSV ASVKLAGNS SLCPVSGWAI YSKDNSVRIG SKGDVFVIRE PFISCSPLEC RTFFLTQGAL LNDKHSNGTI KDRSPYRTLM S CPIGEVPS PYNSRFESVA WSASACHDGT NWLTIGISGP DSGAVAVLKY NGIITDTIKS WRNKILRTQE SECACVNGSC FT IMTDGPS DGQASYKIFR IEKGKIIKSV EMKAPNYHYE ECSCYPDSSE ITCVCRDNWH GSNRPWVSFN QNLEYQMGYI CSG VFGDNP RPNDKTGSCG PVSSNGANGV KGFSFKYGNG VWIGRTKSIS SRKGFEMIWD PNGWTGTDNK FSKKQDIVGI NEWS GYSGS FVQHPELTGL NCIRPCFWVE LIRGRPEENT IWTSGSSISF CGVDSDIVGW SWPDGAELPF TIDK

UniProtKB: Neuraminidase

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Macromolecule #2: ZANAMIVIR

MacromoleculeName: ZANAMIVIR / type: ligand / ID: 2 / Number of copies: 4 / Formula: ZMR
Molecular weightTheoretical: 332.31 Da
Chemical component information

ChemComp-ZMR:
ZANAMIVIR / medication, antivirus, inhibitor*YM

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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: 12 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

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

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Macromolecule #4: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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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

Concentration0.90 mg/mL
BufferpH: 7
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Details: 5 mA
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 288 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Digitization - Frames/image: 1-60 / Number real images: 2889 / Average electron dose: 52.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 48924 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.9000000000000001 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 421625
Startup modelType of model: INSILICO MODEL / In silico model: insilico model generated with cryoSPARC
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.48 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 5) / Number images used: 207640
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: CTFFIND (ver. 4.1.14)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 4.5.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3nss


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9tq8:
Neuraminidase NA isolated from the H1N1 strain A/Victoria/2570/2019 propagated in eggs in complex with zanamivir

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