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- EMDB-52254: Influenza Neuraminidase in complex with N-Acyl Oseltamivir inhibitor -

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Basic information

Entry
Database: EMDB / ID: EMD-52254
TitleInfluenza Neuraminidase in complex with N-Acyl Oseltamivir inhibitor
Map data
Sample
  • Complex: Influenza Neuraminidase in complex with N-Acyl Oseltamivir inhibitor
    • Protein or peptide: Neuraminidase
  • Ligand: (3~{S},4~{R},5~{R},6~{R})-4-acetamido-3-azanyl-5-pentan-3-yloxy-6-(pent-4-ynoylamino)cyclohexene-1-carboxylic acid
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CALCIUM ION
  • Ligand: water
KeywordsInfluenza / Neuraminidase / VIRAL PROTEIN
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
Methodsingle particle reconstruction / cryo EM / Resolution: 2.25 Å
AuthorsMoran E / Davies G
Funding supportEuropean Union, 1 items
OrganizationGrant numberCountry
European Research Council (ERC)European Union
CitationJournal: Proc Natl Acad Sci U S A / Year: 2026
Title: Oseltamivir aziridines are potent influenza neuraminidase inhibitors and imaging agents.
Authors: Merijn B L Vriends / Elisha Moran / Martín Calvelo / Thomas Hansen / Isabelle B Pickles / Xincheng Xin / Marieke Biezeno / Zachary W B Armstrong / Maria J Ferraz / Lei Li / Alice Lilley / ...Authors: Merijn B L Vriends / Elisha Moran / Martín Calvelo / Thomas Hansen / Isabelle B Pickles / Xincheng Xin / Marieke Biezeno / Zachary W B Armstrong / Maria J Ferraz / Lei Li / Alice Lilley / Ruth Harvey / Dmitri V Filippov / Qinghua Liao / Sybrin P Schröder / Gijsbert A van der Marel / Marta Artola / Johannes M F G Aerts / James N Blaza / Jeroen D C Codée / Carme Rovira / Herman S Overkleeft / Gideon J Davies /
Abstract: Influenza neuraminidase (NA) is a critical target for seasonal and pandemic antivirals, including the strains of current concern. Current treatments, such as Zanamivir and Oseltamivir, are limited by ...Influenza neuraminidase (NA) is a critical target for seasonal and pandemic antivirals, including the strains of current concern. Current treatments, such as Zanamivir and Oseltamivir, are limited by noncovalent binding and emerging resistance. We hypothesized that Oseltamivir aziridines would unite transition-state mimicry for tight binding, with aziridine-enabled covalent capture of the catalytic tyrosine, thereby supporting both therapy and activity-based quantification. Here, we present oseltamivir-based aziridines, inspired by cyclophellitol chemistry, that act as covalent inhibitors and activity-based probes via an -acylaziridine warhead. Free-energy calculations, and NMR observations, indicate a H half-chair preference consistent with the NA transition state, and selected analogues inhibit multiple NA subtypes with low nanomolar binding constants. Diverse evidence establishes covalency: time-dependent inactivation, inhibitor washout, intact-mass shifts, MS/MS identification of a tyrosine adduct, and QM/MM reaction profiles, while cryoEM of N1 aligns with the proposed binding mode, revealing an elimination product. The inhibitors demonstrate formidable activity against diverse viral neuraminidases, including H5N1, and further enable imaging and quantification of active NA. With their dual therapeutic and diagnostic potential, these first-in-class inhibitors indeed benefit from transition state mimicry and covalency, and thus offer a powerful platform for antiviral development and neuraminidase imaging, addressing urgent global health needs in influenza treatment and prevention.
History
DepositionDec 4, 2024-
Header (metadata) releaseDec 17, 2025-
Map releaseDec 17, 2025-
UpdateApr 1, 2026-
Current statusApr 1, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_52254.png

Downloads & links

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Map

FileDownload / File: emd_52254.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.9 Å/pix.
x 200 pix.
= 180. Å		0.9 Å/pix.
x 200 pix.
= 180. Å		0.9 Å/pix.
x 200 pix.
= 180. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.9 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0345
Minimum - Maximum-0.1496158 - 0.24152337
Average (Standard dev.)0.00015974556 (±0.01217633)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 180.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_52254_msk_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_52254_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_52254_half_map_2.map
Projections & Slices
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Sample components

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Entire : Influenza Neuraminidase in complex with N-Acyl Oseltamivir inhibitor

EntireName: Influenza Neuraminidase in complex with N-Acyl Oseltamivir inhibitor
Components
  • Complex: Influenza Neuraminidase in complex with N-Acyl Oseltamivir inhibitor
    • Protein or peptide: Neuraminidase
  • Ligand: (3~{S},4~{R},5~{R},6~{R})-4-acetamido-3-azanyl-5-pentan-3-yloxy-6-(pent-4-ynoylamino)cyclohexene-1-carboxylic acid
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CALCIUM ION
  • Ligand: water

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Supramolecule #1: Influenza Neuraminidase in complex with N-Acyl Oseltamivir inhibitor

SupramoleculeName: Influenza Neuraminidase in complex with N-Acyl Oseltamivir inhibitor
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Influenza A virus

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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
Molecular weightTheoretical: 42.499523 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: VKLAGNSSLC PVSGWAPLSK DNSVRIGSKG DVFVIREPFI SCSPLECRTF FLTQGALLND KHSNGTIKDR SPYRTLMSVP IGSVPSPYN ARFESIAWSA SACHDGINWL TIGITGPDNG AVAILKYNGI ITDTIKSWRN NILRTQESEC ACVNGSCFTV M TDGPSNGQ ...String:
VKLAGNSSLC PVSGWAPLSK DNSVRIGSKG DVFVIREPFI SCSPLECRTF FLTQGALLND KHSNGTIKDR SPYRTLMSVP IGSVPSPYN ARFESIAWSA SACHDGINWL TIGITGPDNG AVAILKYNGI ITDTIKSWRN NILRTQESEC ACVNGSCFTV M TDGPSNGQ ASYKIFRIEK GKIVKSVEMN APNYHYEECS CYPDSSEITC VCRDNWHGSN RPWVSFNQNL EYQIGYICSG IF GDNPRPN DKTGSCGPVS SNGANGVKGF SFKYGNGVWI GRTKSISSRN GFEMIWDPNG WTGTDNNFSI KQDIVGINEW SGY SGSFVM HPELTGLDCI VPCFWVELIR GRPKENTIWT SGSSISFCGV NSDTVGWSWP DGAELPFTID K

UniProtKB: Neuraminidase

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Macromolecule #2: (3~{S},4~{R},5~{R},6~{R})-4-acetamido-3-azanyl-5-pentan-3-yloxy-6...

MacromoleculeName: (3~{S},4~{R},5~{R},6~{R})-4-acetamido-3-azanyl-5-pentan-3-yloxy-6-(pent-4-ynoylamino)cyclohexene-1-carboxylic acid
type: ligand / ID: 2 / Number of copies: 4 / Formula: A1IVW
Molecular weightTheoretical: 379.451 Da

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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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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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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 392 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration1.5 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
25.0 mMTrisTris
150.0 mMNaClSodium chloride
100.0 mMCaCl2Calcium chloride

Details: 25 mM Tris pH 7.0 150 mM NaCl, 100 mM CaCl2
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 180 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: Blot force -10 and blot time 3 seconds.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 43.99 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.4000000000000001 µm / Nominal defocus min: 0.6 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.25 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 5.0) / Number images used: 229896
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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