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- PDB-6t0w: Human Influenza B polymerase recycling complex -

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Basic information

Entry
Database: PDB / ID: 6t0w
TitleHuman Influenza B polymerase recycling complex
Components
  • Polymerase PB2
  • Polymerase acidic protein
  • RNA-directed RNA polymerase catalytic subunit
  • vRNAVault RNA
KeywordsVIRAL PROTEIN / Influenza / polymerase / viral transcription / RNA
Function / homology
Function and homology information


cap snatching / suppression by virus of host RNA polymerase II activity / 7-methylguanosine mRNA capping / viral transcription / virion / viral RNA genome replication / RNA-directed RNA polymerase / host cell cytoplasm / endonuclease activity / RNA-directed 5'-3' RNA polymerase activity ...cap snatching / suppression by virus of host RNA polymerase II activity / 7-methylguanosine mRNA capping / viral transcription / virion / viral RNA genome replication / RNA-directed RNA polymerase / host cell cytoplasm / endonuclease activity / RNA-directed 5'-3' RNA polymerase activity / transcription, DNA-templated / Hydrolases; Acting on ester bonds / nucleotide binding / host cell nucleus / RNA binding / metal ion binding
Influenza RNA-dependent RNA polymerase subunit PB2 / Influenza RNA-dependent RNA polymerase subunit PA / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PA / PA/PA-X superfamily / Polymerase acidic protein / PB2, C-terminal / RNA-directed RNA polymerase, negative-strand RNA virus / Influenza RNA-dependent RNA polymerase subunit PB2 / Influenza RNA-dependent RNA polymerase subunit PB1
Polymerase basic protein 2 / Polymerase basic protein 2 / RNA-directed RNA polymerase catalytic subunit / Polymerase acidic protein
Biological speciesInfluenza B virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.18 Å
AuthorsWandzik, J.M. / Kouba, T. / Karuppasamy, M. / 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.
Validation Report
SummaryFull reportAbout validation report
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

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Structure visualization

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Assembly

Deposited unit
A: Polymerase acidic protein
B: RNA-directed RNA polymerase catalytic subunit
C: Polymerase PB2
V: vRNA


Theoretical massNumber of molelcules
Total (without water)273,8404
Polymers273,8404
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area26230 Å2
ΔGint-172 kcal/mol
Surface area53770 Å2
MethodPISA

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Components

#1: Protein Polymerase acidic protein


Mass: 85822.781 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza B virus / Gene: PA / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: Q5V8Z9, 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: 86207.016 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza B virus / Gene: PB1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q5V8Y6, RNA-directed RNA polymerase
#3: Protein Polymerase PB2


Mass: 90971.281 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza B virus / Gene: PB2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A0A4Y5WMY1, UniProt: Q5V8X3*PLUS
#4: RNA chain vRNA / Vault RNA


Mass: 10838.431 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Influenza B virus

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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
1Human Influenza B polymerase recycling complexCOMPLEX#1-#40MULTIPLE SOURCES
2PolymeraseCOMPLEX#1-#31RECOMBINANT
3Nucleic acidCOMPLEX#41RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Influenza B virus11520
23Influenza B virus11520
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 FIELDBright-field microscopy
Image recordingElectron dose: 35 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: 3.18 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 68333 / Symmetry type: POINT
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00810820
ELECTRON MICROSCOPYf_angle_d0.77814714
ELECTRON MICROSCOPYf_dihedral_angle_d11.5276515
ELECTRON MICROSCOPYf_chiral_restr0.0511637
ELECTRON MICROSCOPYf_plane_restr0.0071796

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