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- PDB-6t0r: Bat Influenza A polymerase product dissociation complex using 44-... -

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Basic information

Entry
Database: PDB / ID: 6t0r
TitleBat Influenza A polymerase product dissociation complex using 44-mer vRNA template with mutated oligo(U) sequence
Components
  • Polymerase acidic protein
  • Polymerase basic protein 2
  • RNA-directed RNA polymerase catalytic subunit
  • vRNA
KeywordsVIRAL PROTEIN / Influenza / polymerase / viral transcription / RNA
Function / homology
Function and homology information


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 / virion component / host cell cytoplasm / RNA-directed RNA polymerase / viral translational frameshifting / viral RNA genome replication ...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 / virion component / host cell cytoplasm / RNA-directed RNA polymerase / viral translational frameshifting / viral RNA genome replication / RNA-dependent RNA polymerase activity / nucleotide binding / DNA-templated transcription / host cell nucleus / RNA binding / metal ion binding / cytoplasm
Similarity search - Function
Influenza RNA-dependent RNA polymerase subunit PA, endonuclease domain / Restriction Endonuclease / 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-dependent RNA polymerase subunit PA, endonuclease domain / Restriction Endonuclease / 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 / 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. / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
RNA / RNA (> 10) / Polymerase basic protein 2 / RNA-directed RNA polymerase catalytic subunit / Polymerase acidic protein
Similarity search - Component
Biological speciesInfluenza A virus
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.82 Å
AuthorsWandzik, J.M. / Kouba, T. / Cusack, S.
Funding support France, 1items
OrganizationGrant numberCountry
French National Research AgencyANR-18-CE11-0028 France
CitationJournal: Cell / Year: 2020
Title: A Structure-Based Model for the Complete Transcription Cycle of Influenza Polymerase.
Authors: Joanna M Wandzik / Tomas Kouba / Manikandan Karuppasamy / Alexander Pflug / Petra Drncova / Jan Provaznik / Nayara Azevedo / Stephen Cusack /
Abstract: Influenza polymerase uses unique mechanisms to synthesize capped and polyadenylated mRNAs from the genomic viral RNA (vRNA) template, which is packaged inside ribonucleoprotein particles (vRNPs). ...Influenza polymerase uses unique mechanisms to synthesize capped and polyadenylated mRNAs from the genomic viral RNA (vRNA) template, which is packaged inside ribonucleoprotein particles (vRNPs). Here, we visualize by cryoelectron microscopy the conformational dynamics of the polymerase during the complete transcription cycle from pre-initiation to termination, focusing on the template trajectory. After exiting the active site cavity, the template 3' extremity rebinds into a specific site on the polymerase surface. Here, it remains sequestered during all subsequent transcription steps, forcing the template to loop out as it further translocates. At termination, the strained connection between the bound template 5' end and the active site results in polyadenylation by stuttering at uridine 17. Upon product dissociation, further conformational changes release the trapped template, allowing recycling back into the pre-initiation state. Influenza polymerase thus performs transcription while tightly binding to and protecting both template ends, allowing efficient production of multiple mRNAs from a single vRNP.
History
DepositionOct 3, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 15, 2020Provider: repository / Type: Initial release
Revision 1.1Apr 29, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2May 27, 2020Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.3May 22, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: Polymerase acidic protein
B: RNA-directed RNA polymerase catalytic subunit
C: Polymerase basic protein 2
V: vRNA


Theoretical massNumber of molelcules
Total (without water)278,4674
Polymers278,4674
Non-polymers00
Water55831
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area38890 Å2
ΔGint-229 kcal/mol
Surface area67550 Å2
MethodPISA

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Components

#1: Protein Polymerase acidic protein


Mass: 85490.930 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (A/little yellow-shouldered bat/Guatemala/060/2010(H17N10))
Strain: A/little yellow-shouldered bat/Guatemala/060/2010(H17N10)
Gene: PA / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: H6QM92
#2: Protein RNA-directed RNA polymerase catalytic subunit


Mass: 87936.312 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (A/little yellow-shouldered bat/Guatemala/060/2010(H17N10))
Strain: A/little yellow-shouldered bat/Guatemala/060/2010(H17N10)
Gene: PB1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: H6QM91, RNA-directed RNA polymerase
#3: Protein Polymerase basic protein 2


Mass: 91027.141 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (A/little yellow-shouldered bat/Guatemala/060/2010(H17N10))
Strain: A/little yellow-shouldered bat/Guatemala/060/2010(H17N10)
Gene: PB2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: H6QM90
#4: RNA chain vRNA


Mass: 14012.289 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 31 / Source method: isolated from a natural source / Formula: H2O

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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

Component
IDNameTypeEntity IDParent-IDSource
1Bat Influenza A polymerase product dissociation complex using 44-mer vRNA template with mutated oligo(U) sequenceCOMPLEX#1-#40MULTIPLE SOURCES
2PolymeraseCOMPLEX#1-#31RECOMBINANT
3Nucleic acidCOMPLEX#41RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Influenza A virus (A/little yellow-shouldered bat/Guatemala/060/2010(H17N10))1129347
23synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Trichoplusia ni (cabbage looper)7111
23synthetic construct (others)32630
Buffer solutionpH: 7.5
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: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 39 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 2.82 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 52294 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00514421
ELECTRON MICROSCOPYf_angle_d0.62519536
ELECTRON MICROSCOPYf_dihedral_angle_d13.9728744
ELECTRON MICROSCOPYf_chiral_restr0.0452141
ELECTRON MICROSCOPYf_plane_restr0.0062440

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