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- PDB-6kv5: Structure of influenza D virus apo polymerase -

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Basic information

Entry
Database: PDB / ID: 6kv5
TitleStructure of influenza D virus apo polymerase
Components
  • Polymerase 3
  • Polymerase PB2
  • RNA-directed RNA polymerase catalytic subunit
KeywordsVIRAL PROTEIN / influenza D virus / polymerase / cryo-EM
Function / homology
Function and homology information


cap snatching / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / 7-methylguanosine mRNA capping / host cell mitochondrion / virion component / symbiont-mediated suppression of host gene expression / RNA-directed RNA polymerase / viral RNA genome replication / nucleotide binding / RNA-directed RNA polymerase activity ...cap snatching / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / 7-methylguanosine mRNA capping / host cell mitochondrion / virion component / symbiont-mediated suppression of host gene expression / RNA-directed RNA polymerase / viral RNA genome replication / nucleotide binding / RNA-directed RNA polymerase activity / DNA-templated transcription / host cell nucleus / RNA binding
Similarity search - Function
RNA-dependent RNA polymerase, bunyaviral / Bunyavirus RNA dependent RNA polymerase / 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 ...RNA-dependent RNA polymerase, bunyaviral / Bunyavirus RNA dependent RNA polymerase / 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 / 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-directed RNA polymerase catalytic subunit / Polymerase basic protein 2 / Polymerase 3
Similarity search - Component
Biological speciesInfluenza D virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsPeng, Q. / Peng, R. / Qi, J. / Gao, G.F. / Shi, Y.
Funding support China, 1items
OrganizationGrant numberCountry
Chinese Academy of SciencesXDB08020100 China
Citation
Journal: Nat Microbiol / Year: 2019
Title: Structural insight into RNA synthesis by influenza D polymerase.
Authors: Qi Peng / Yuqian Liu / Ruchao Peng / Min Wang / Wei Yang / Hao Song / Yuhai Chen / Sheng Liu / Min Han / Xinzheng Zhang / Peiyi Wang / Jinghua Yan / Buchang Zhang / Jianxun Qi / Tao Deng / ...Authors: Qi Peng / Yuqian Liu / Ruchao Peng / Min Wang / Wei Yang / Hao Song / Yuhai Chen / Sheng Liu / Min Han / Xinzheng Zhang / Peiyi Wang / Jinghua Yan / Buchang Zhang / Jianxun Qi / Tao Deng / George F Gao / Yi Shi /
Abstract: The influenza virus polymerase uses capped RNA primers to initiate transcription, and a combination of terminal and internal de novo initiations for the two-step replication process by binding the ...The influenza virus polymerase uses capped RNA primers to initiate transcription, and a combination of terminal and internal de novo initiations for the two-step replication process by binding the conserved viral genomic RNA (vRNA) or complementary RNA (cRNA) promoter. Here, we determined the apo and promoter-bound influenza D polymerase structures using cryo-electron microscopy and found the polymerase has an evolutionarily conserved stable core structure with inherently flexible peripheral domains. Strikingly, two conformations (mode A and B) of the vRNA promoter were observed where the 3'-vRNA end can bind at two different sites, whereas the cRNA promoter only binds in the mode B conformation. Functional studies confirmed the critical role of the mode B conformation for vRNA synthesis via the intermediate cRNA but not for cRNA production, which is mainly regulated by the mode A conformation. Both conformations participate in the regulation of the transcription process. This work advances our understanding of the regulatory mechanisms for the synthesis of different RNA species by influenza virus polymerase and opens new opportunities for antiviral drug design.
#1: Journal: Nat Microbiol / Year: 2019
Title: Structural insight into RNA synthesis by influenza D polymerase.
Authors: Qi Peng / Yuqian Liu / Ruchao Peng / Min Wang / Wei Yang / Hao Song / Yuhai Chen / Sheng Liu / Min Han / Xinzheng Zhang / Peiyi Wang / Jinghua Yan / Buchang Zhang / Jianxun Qi / Tao Deng / ...Authors: Qi Peng / Yuqian Liu / Ruchao Peng / Min Wang / Wei Yang / Hao Song / Yuhai Chen / Sheng Liu / Min Han / Xinzheng Zhang / Peiyi Wang / Jinghua Yan / Buchang Zhang / Jianxun Qi / Tao Deng / George F Gao / Yi Shi /
Abstract: The influenza virus polymerase uses capped RNA primers to initiate transcription, and a combination of terminal and internal de novo initiations for the two-step replication process by binding the ...The influenza virus polymerase uses capped RNA primers to initiate transcription, and a combination of terminal and internal de novo initiations for the two-step replication process by binding the conserved viral genomic RNA (vRNA) or complementary RNA (cRNA) promoter. Here, we determined the apo and promoter-bound influenza D polymerase structures using cryo-electron microscopy and found the polymerase has an evolutionarily conserved stable core structure with inherently flexible peripheral domains. Strikingly, two conformations (mode A and B) of the vRNA promoter were observed where the 3'-vRNA end can bind at two different sites, whereas the cRNA promoter only binds in the mode B conformation. Functional studies confirmed the critical role of the mode B conformation for vRNA synthesis via the intermediate cRNA but not for cRNA production, which is mainly regulated by the mode A conformation. Both conformations participate in the regulation of the transcription process. This work advances our understanding of the regulatory mechanisms for the synthesis of different RNA species by influenza virus polymerase and opens new opportunities for antiviral drug design.
History
DepositionSep 3, 2019Deposition site: PDBJ / Processing site: PDBJ
SupersessionOct 2, 2019ID: 6AB7
Revision 1.0Oct 2, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 6, 2019Group: Data collection / Other / Category: cell / Item: _cell.Z_PDB
Revision 1.2Apr 14, 2021Group: 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
Revision 1.3Mar 27, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: Polymerase 3
B: RNA-directed RNA polymerase catalytic subunit
C: Polymerase PB2


Theoretical massNumber of molelcules
Total (without water)257,6553
Polymers257,6553
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, homology
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Polymerase 3 / P3 subunit of influenza D virus polymerase


Mass: 83036.086 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza D virus (D/swine/Oklahoma/1334/2011)
Strain: D/swine/Oklahoma/1334/2011 / Gene: P3
Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
References: UniProt: K9LHJ4
#2: Protein RNA-directed RNA polymerase catalytic subunit / PB1 subunit of influenza D virus polymerase


Mass: 86138.844 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza D virus (D/swine/Oklahoma/1334/2011)
Strain: D/swine/Oklahoma/1334/2011 / Gene: PB1
Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
References: UniProt: K9LH03, RNA-directed RNA polymerase
#3: Protein Polymerase PB2 / PB2 subunit of influenza D virus polymerase


Mass: 88480.484 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza D virus (D/swine/Oklahoma/1334/2011)
Strain: D/swine/Oklahoma/1334/2011 / Gene: PB2
Production host: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
References: UniProt: K9LHF3

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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 D virus polymerase complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.25 MDa / Experimental value: YES
Source (natural)Organism: Influenza D virus
Source (recombinant)Organism: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4GctfCTF correction
7UCSF Chimeramodel fitting
9RELION2initial Euler assignment
10RELION2final Euler assignment
11RELION2classification
12RELION23D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 108120 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 5D98

5d98


Accession code: 5D98 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00413795
ELECTRON MICROSCOPYf_angle_d0.81318529
ELECTRON MICROSCOPYf_dihedral_angle_d12.6888466
ELECTRON MICROSCOPYf_chiral_restr0.0512007
ELECTRON MICROSCOPYf_plane_restr0.0062364

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