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- PDB-6tw1: Bat Influenza A polymerase termination complex with pyrophosphate... -

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Basic information

Entry
Database: PDB / ID: 6tw1
TitleBat Influenza A polymerase termination complex with pyrophosphate using 44-mer vRNA template with mutated oligo(U) sequence
Components
  • Polymerase acidic protein
  • Polymerase basic protein 2
  • RNA-directed RNA polymerase catalytic subunit
  • mRNAMessenger RNA
  • 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 / host cell mitochondrion / 7-methylguanosine mRNA capping / viral transcription / virion / RNA-directed RNA polymerase / viral RNA genome replication / RNA-directed 5'-3' RNA polymerase activity / transcription, DNA-templated ...cap snatching / suppression by virus of host RNA polymerase II activity / host cell mitochondrion / 7-methylguanosine mRNA capping / viral transcription / virion / RNA-directed RNA polymerase / viral RNA genome replication / RNA-directed 5'-3' RNA polymerase activity / transcription, DNA-templated / host cell cytoplasm / host cell nucleus / RNA binding / metal ion binding / nucleus / cytoplasm
PA/PA-X superfamily / 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 / Influenza RNA-dependent RNA polymerase subunit PA / Influenza RNA-dependent RNA polymerase subunit PA, endonuclease domain / Restriction Endonuclease / 3-Layer(aba) Sandwich / Alpha Beta
Polymerase basic protein 2 / RNA-directed RNA polymerase catalytic subunit / Polymerase acidic protein
Biological speciesInfluenza A virus
Influenza B virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 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.
Validation Report
SummaryFull reportAbout validation report
History
DepositionJan 11, 2020Deposition 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

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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 basic protein 2
V: vRNA
M: mRNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)282,27711
Polymers281,6515
Non-polymers6276
Water2,414134
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area48010 Å2
ΔGint-296 kcal/mol
Surface area86550 Å2
MethodPISA

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Components

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Protein , 3 types, 3 molecules ABC

#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

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RNA chain , 2 types, 2 molecules VM

#4: RNA chain vRNA / Vault RNA


Mass: 14012.289 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Influenza B virus
#5: RNA chain mRNA / Messenger RNA


Mass: 3183.988 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Influenza B virus

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Non-polymers , 4 types, 140 molecules

#6: Chemical ChemComp-M4H / 5-oxidanyl-4-oxidanylidene-1-[(1-pyrrolo[2,3-b]pyridin-1-ylcyclopentyl)methyl]pyridine-3-carboxylic acid


Mass: 353.372 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C19H19N3O4
#7: Chemical
ChemComp-MG / MAGNESIUM ION / Magnesium


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
#8: Chemical ChemComp-POP / PYROPHOSPHATE 2- / Pyrophosphate


Mass: 175.959 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: H2O7P2
#9: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 134 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN

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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 termination complex with pyrophosphate using 44-mer vRNA template with mutated oligo(U) sequenceCOMPLEX#1-#50MULTIPLE SOURCES
2PolymeraseCOMPLEX#1-#31RECOMBINANT
3Nucleic acidsNucleic acidCOMPLEX#4-#51RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Influenza A virus (A/little yellow-shouldered bat/Guatemala/060/2010(H17N10))1129347
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: 46 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 101612 / Symmetry type: POINT
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00818935
ELECTRON MICROSCOPYf_angle_d0.79825747
ELECTRON MICROSCOPYf_dihedral_angle_d19.1067336
ELECTRON MICROSCOPYf_chiral_restr0.0522867
ELECTRON MICROSCOPYf_plane_restr0.0063153

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