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- PDB-9lck: Cryo-EM structure of influenza polymerase bound to cRNA promoter -

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Basic information

Entry
Database: PDB / ID: 9lck
TitleCryo-EM structure of influenza polymerase bound to cRNA promoter
Components
  • 3'-cRNA
  • 5'-cRNA
  • PB2-S1
  • Polymerase acidic protein
  • RNA-directed RNA polymerase catalytic subunit
KeywordsVIRAL PROTEIN/RNA / influenza polymerase / VIRAL PROTEIN-RNA complex
Function / homology
Function and homology information


host cell cytosol / cap snatching / viral transcription / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / host cell mitochondrion / endonuclease activity / Hydrolases; Acting on ester bonds / host cell cytoplasm / symbiont-mediated suppression of host gene expression / viral translational frameshifting ...host cell cytosol / cap snatching / viral transcription / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / host cell mitochondrion / endonuclease activity / Hydrolases; Acting on ester bonds / host cell cytoplasm / symbiont-mediated suppression of host gene expression / viral translational frameshifting / RNA-directed RNA polymerase / viral RNA genome replication / hydrolase activity / nucleotide binding / RNA-directed RNA polymerase activity / host cell nucleus / DNA-templated transcription / RNA binding / metal ion binding / identical protein binding
Similarity search - Function
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 CAP binding domain ...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 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
RNA / RNA (> 10) / PB2-S1 / Polymerase acidic protein / RNA-directed RNA polymerase catalytic subunit
Similarity search - Component
Biological speciesInfluenza A virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.98 Å
AuthorsWu, Y.X. / Liang, H.H.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Commun Biol / Year: 2025
Title: Cryo-EM structure of influenza polymerase bound to the cRNA promoter provides insights into the mechanism of viral replication.
Authors: Yixi Wu / Minke Li / Huanhuan Li / Yifan Bai / Tianli Sun / Shaohui Huang / Yingfang Liu / Huanhuan Liang /
Abstract: Influenza virus polymerase (FluPol) synthesizes the complementary RNA (cRNA) and the viral RNA (vRNA) using distinct de novo initiation strategies during genome replication, known as internal and ...Influenza virus polymerase (FluPol) synthesizes the complementary RNA (cRNA) and the viral RNA (vRNA) using distinct de novo initiation strategies during genome replication, known as internal and terminal initiation, respectively. The de novo initiation mechanisms, especially the internal initiation process, which includes a template realignment process, are still not well understood. Here, we present a cryo-electron microscopy structure of H5N1 FluPol bound to the cRNA promoter. In combination with structural analyses and structure-guided mutagenesis studies, we identified several previously unreported structural features of FluPol essential for internal initiation. The B loop adopts an "open" conformation, allowing translocation of the 3' terminus of cRNA (3'-cRNA) template into the catalytic cavity. The dynamic of incoming 3'-cRNA template is limited by the priming and realignment loop (PR loop), which facilitates the cRNA template realignment process. An asparagine cluster above the catalytic cavity is required for polymerase activity. Our findings provide structural insights into the mechanism of replication internal initiation of FluPol.
History
DepositionJan 4, 2025Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Jan 21, 2026Provider: repository / Type: Initial release
Revision 1.1Aug 5, 2026Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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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: PB2-S1
D: 3'-cRNA
E: 5'-cRNA


Theoretical massNumber of molelcules
Total (without water)182,7845
Polymers182,7845
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Polymerase acidic protein / RNA-directed RNA polymerase subunit P2


Mass: 82669.469 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (strain A/Goose/Guangdong/1/1996 H5N1 genotype Gs/Gd)
Gene: PA
Production host: Insect cell expression vector pTIE1 (others)
References: UniProt: Q9Q0U9, 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: 86492.195 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (strain A/Goose/Guangdong/1/1996 H5N1 genotype Gs/Gd)
Gene: PB1
Production host: Insect cell expression vector pTIE1 (others)
References: UniProt: Q9Q0V0, RNA-directed RNA polymerase
#3: Protein/peptide PB2-S1 / Polymerase PB2


Mass: 5009.662 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (strain A/Goose/Guangdong/1/1996 H5N1 genotype Gs/Gd)
Gene: PB2
Production host: Insect cell expression vector pTIE1 (others)
References: UniProt: P0DOG5
#4: RNA chain 3'-cRNA


Mass: 2775.719 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Influenza A virus (strain A/Goose/Guangdong/1/1996 H5N1 genotype Gs/Gd)
#5: RNA chain 5'-cRNA


Mass: 5836.540 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Influenza A virus (strain A/Goose/Guangdong/1/1996 H5N1 genotype Gs/Gd)
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: influenza polymerase / Type: COMPLEX / Entity ID: #1-#2, #4-#5, #3 / Source: RECOMBINANT
Source (natural)Organism: Influenza A virus (strain A/Goose/Guangdong/1/1996 H5N1 genotype Gs/Gd)
Source (recombinant)Organism: Insect cell expression vector pTIE1 (others)
Buffer solutionpH: 7.8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3500 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: DIRECT ELECTRON DE-16 (4k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487 / Category: model refinement
CTF correctionType: NONE
3D reconstructionResolution: 2.98 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 144735 / Symmetry type: POINT
RefinementHighest resolution: 2.98 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00410192
ELECTRON MICROSCOPYf_angle_d0.72713883
ELECTRON MICROSCOPYf_dihedral_angle_d8.0531581
ELECTRON MICROSCOPYf_chiral_restr0.0411538
ELECTRON MICROSCOPYf_plane_restr0.0051688

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