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- PDB-9ryc: Structure of human 1918 influenza A polymerase heterotrimer in co... -

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Basic information

Entry
Database: PDB / ID: 9ryc
TitleStructure of human 1918 influenza A polymerase heterotrimer in complex with WSN NEP.
Components
  • Nuclear export protein
  • Polymerase acidic protein
  • Polymerase basic protein 2
  • RNA-directed RNA polymerase catalytic subunit
KeywordsVIRAL PROTEIN / influenza virus / nuclear export / NEP / NS2 / 1918 polymerase / spanish Flu / viral polymerase / cryo-EM
Function / homology
Function and homology information


exit of virus from host cell nucleus through nuclear pore / cap snatching / viral transcription / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / host cell mitochondrion / 7-methylguanosine mRNA capping / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / virion component / endonuclease activity / Hydrolases; Acting on ester bonds ...exit of virus from host cell nucleus through nuclear pore / cap snatching / viral transcription / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / host cell mitochondrion / 7-methylguanosine mRNA capping / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / virion component / endonuclease activity / Hydrolases; Acting on ester bonds / host cell cytoplasm / symbiont-mediated suppression of host gene expression / hydrolase activity / viral translational frameshifting / RNA-directed RNA polymerase / viral RNA genome replication / nucleotide binding / RNA-directed RNA polymerase activity / DNA-templated transcription / host cell nucleus / RNA binding / metal ion binding
Similarity search - Function
Influenza nuclear export protein NS2 / Influenza non-structural protein (NS2) / Influenza RNA-dependent RNA polymerase subunit PB2 / PB2, C-terminal / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB1 / : / : / Influenza RNA polymerase PB2 N-terminal region / Influenza RNA polymerase PB2 second domain ...Influenza nuclear export protein NS2 / Influenza non-structural protein (NS2) / Influenza RNA-dependent RNA polymerase subunit PB2 / PB2, C-terminal / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB1 / : / : / Influenza RNA polymerase PB2 N-terminal region / Influenza RNA polymerase PB2 second domain / : / : / Influenza RNA polymerase PB2 middle domain / Influenza RNA polymerase PB2 C-terminal domain / : / Influenza RNA polymerase PB2 6th domain / : / Influenza RNA polymerase PB2 CAP binding domain / : / Influenza RNA polymerase PB2 helical domain / Polymerase acidic protein / Influenza RNA-dependent RNA polymerase subunit PA / Influenza RNA-dependent RNA polymerase subunit PA, endonuclease domain / Influenza RNA-dependent RNA polymerase subunit PA / RNA-directed RNA polymerase, negative-strand RNA virus / RdRp of negative ssRNA viruses with segmented genomes catalytic domain profile.
Similarity search - Domain/homology
Polymerase acidic protein / RNA-directed RNA polymerase catalytic subunit / Polymerase basic protein 2 / Nuclear export protein
Similarity search - Component
Biological speciesInfluenza A virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.93 Å
AuthorsRep, A. / Wang, F. / Chen, K. / Carrique, L. / Grimes, J.M. / Fodor, E.
Funding support United Kingdom, 8items
OrganizationGrant numberCountry
Wellcome Trust200835/Z/16/Z United Kingdom
Wellcome Trust222510/Z/21/Z United Kingdom
Medical Research Council (MRC, United Kingdom)MR/R009945/1 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/X008312/1 United Kingdom
Wellcome Trust093305/Z/10/Z United Kingdom
Wellcome Trust060208/Z/00/Z United Kingdom
Wellcome Trust203141/Z/16/Z United Kingdom
Wellcome Trust227570/Z/23/Z United Kingdom
CitationJournal: Sci Adv / Year: 2026
Title: Regulatory hotspot on the influenza A virus polymerase revealed through the structure of the NEP-polymerase complex.
Authors: Alison Rep / Fangzheng Wang / Kuang-Yu Chen / Loïc Carrique / Jane Sharps / Jonathan M Grimes / Ervin Fodor /
Abstract: Influenza A virus (IAV) transcribes and replicates its segmented RNA genome in the host nucleus within viral ribonucleoproteins (vRNPs), which are exported for virion assembly. The nuclear export ...Influenza A virus (IAV) transcribes and replicates its segmented RNA genome in the host nucleus within viral ribonucleoproteins (vRNPs), which are exported for virion assembly. The nuclear export protein (NEP) is essential for this process and also regulates viral RNA synthesis, implicating a direct interaction with the viral RNA polymerase. Here, we present a 2.5-Å cryogenic electron microscopy structure of NEP bound to the IAV polymerase and demonstrate that NEP alone is sufficient to promote vRNP export, with the viral matrix protein 1 enhancing export efficiency. NEP forms a four-helix bundle that binds at the interface of the PA C-terminal domain and PB1 N terminus of the polymerase. The NEP binding site at this interface overlaps with those for the host ANP32 and the C-terminal domain of RNA polymerase II, indicating that it functions as a regulatory hotspot coordinating transitions of the viral polymerase between RNA synthesis and nuclear export, revealing a critical layer of control in the IAV replication cycle.
History
DepositionJul 15, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 24, 2025Provider: repository / Type: Initial release
Revision 1.0Dec 24, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Dec 24, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Dec 24, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 24, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 24, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Dec 24, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Dec 24, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Feb 4, 2026Group: Data collection / Database references / Structure summary
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Revision 1.1Feb 4, 2026Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Polymerase acidic protein
B: RNA-directed RNA polymerase catalytic subunit
C: Polymerase basic protein 2
D: Nuclear export protein


Theoretical massNumber of molelcules
Total (without water)271,4034
Polymers271,4034
Non-polymers00
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 Polymerase acidic protein / RNA-directed RNA polymerase subunit P2


Mass: 82707.391 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (A/Brevig Mission/1/1918(H1N1))
Gene: PA / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: Q3HM39, Hydrolases; Acting on ester bonds
#2: Protein RNA-directed RNA polymerase catalytic subunit / Polymerase basic protein 1 / PB1 / RNA-directed RNA polymerase subunit P1


Mass: 86625.211 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (A/Brevig Mission/1/1918(H1N1))
Gene: PB1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q3HM40, RNA-directed RNA polymerase
#3: Protein Polymerase basic protein 2


Mass: 86849.133 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: PB2 plus the remaining TEV cleavage site
Source: (gene. exp.) Influenza A virus (A/Brevig Mission/1/1918(H1N1))
Gene: PB2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q3HM41
#4: Protein Nuclear export protein / NEP / Non-structural protein 2 / NS2


Mass: 15221.367 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: GST-3C-NEP fusion protein
Source: (gene. exp.) Influenza A virus (A/Brevig Mission/1/1918(H1N1))
Gene: NS / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q77IX1
Has protein modificationN

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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: Structure of human 1918 influenza A polymerase heterotrimer in complex with WSN NEP
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.283 MDa / Experimental value: YES
Source (natural)Organism: Influenza A virus (A/Brevig Mission/1/1918(H1N1))
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
125 mMHEPES1
2150 mMNaCl1
31
SpecimenConc.: 0.33 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 24000 nm / Nominal defocus min: 5000 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: 50 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k)
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.2 - 4.7particle selection
2EPU3.14image acquisition
4cryoSPARC4.2 - 4.7CTF correction
7UCSF ChimeraX1.7model fitting
8Coot0.9.8.3model fitting
10PHENIX1.19.2model refinement
11cryoSPARC4.2 - 4.7initial Euler assignment
12cryoSPARC4.2 - 4.7final Euler assignment
13cryoSPARC4.2 - 4.7classification
14cryoSPARC4.2 - 4.73D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.93 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 35000 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 73.12 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00314784
ELECTRON MICROSCOPYf_angle_d0.484719914
ELECTRON MICROSCOPYf_chiral_restr0.03692172
ELECTRON MICROSCOPYf_plane_restr0.00322565
ELECTRON MICROSCOPYf_dihedral_angle_d4.06311957

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