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- EMDB-56126: Neuraminidase NA isolated from the H1N1 strain A/Victoria/2570/20... -

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Basic information

Entry
Database: EMDB / ID: EMD-56126
TitleNeuraminidase NA isolated from the H1N1 strain A/Victoria/2570/2019 propagated in eggs
Map dataUnsharpened map of full-length vN1 in HDM detergent
Sample
  • Complex: Neuraminidase NA isolated from the H1N1 strain A/Victoria/2570/2019 propagated in eggs
    • Protein or peptide: Neuraminidase
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CALCIUM ION
Keywordsglycosidase / influenza virus / viral release / HYDROLASE
Biological speciesInfluenza A virus (A/(H1N1))
Methodsingle particle reconstruction / cryo EM / Resolution: 3.58 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 3, 2026-
Map releaseJun 3, 2026-
UpdateJun 3, 2026-
Current statusJun 3, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_56126.png

Downloads & links

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Map

FileDownload / File: emd_56126.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened map of full-length vN1 in HDM detergent
Projections & slices

Image control

Size
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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

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.022 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00631
Minimum - Maximum-0.03306992 - 0.047264043
Average (Standard dev.)0.0000060742477 (±0.0012612103)
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_56126_msk_1.map
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Additional map: Unsharpened map of full-length vN1 in HDM detergent...

Fileemd_56126_additional_1.map
AnnotationUnsharpened map of full-length vN1 in HDM detergent (no symmetry applied)
Projections & Slices
AxesZYX

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Half map: Half map 1 of vN1 in HDM detergent...

Fileemd_56126_half_map_1.map
AnnotationHalf map 1 of vN1 in HDM detergent used for refinement and gold-standard FSC resolution calculation
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Histogram

Histogram (log scale)

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

Fileemd_56126_half_map_2.map
AnnotationHalf map 2 of full-length vN1 in HDM detergent used for refinement and gold-standard FSC resolution calculation
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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
Components
  • Complex: Neuraminidase NA isolated from the H1N1 strain A/Victoria/2570/2019 propagated in eggs
    • Protein or peptide: Neuraminidase
  • 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
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

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

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 2 / 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 #3: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 3 / Number of copies: 8 / 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

Concentration0.9 mg/mL
BufferpH: 7
Details: 20 mM HEPES pH 7, 150 mM NaCl, 1 mM CaCl2, 0.0001% HDM
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) / Detector mode: COUNTING / Digitization - Frames/image: 1-60 / Number grids imaged: 1 / Number real images: 2282 / Average electron dose: 57.3 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: 456795
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.14) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL / In silico model: insilico model generated with cryoSPARC
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.58 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 5) / Number images used: 204391
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 4.5.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 5)
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-9tq7:
Neuraminidase NA isolated from the H1N1 strain A/Victoria/2570/2019 propagated in eggs

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