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- PDB-6qcs: Influenza B polymerase pre-initiation complex -

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Basic information

Entry
Database: PDB / ID: 6qcs
TitleInfluenza B polymerase pre-initiation complex
Components
  • 3 end
  • 5 end
  • Capped RNA
  • Polymerase acidic protein
  • Polymerase basic protein 2
  • RNA-directed RNA polymerase catalytic subunit
KeywordsVIRAL PROTEIN / Influenza polzmerase / Viral protein
Function / homology
Function and homology information


cap snatching / suppression by virus of host RNA polymerase II activity / 7-methylguanosine mRNA capping / viral transcription / virion / ec:2.7.7.48: / viral RNA genome replication / endonuclease activity / RNA-directed 5'-3' RNA polymerase activity / host cell cytoplasm ...cap snatching / suppression by virus of host RNA polymerase II activity / 7-methylguanosine mRNA capping / viral transcription / virion / ec:2.7.7.48: / viral RNA genome replication / endonuclease activity / RNA-directed 5'-3' RNA polymerase activity / host cell cytoplasm / ec:3.1.-.-: / transcription, DNA-templated / host cell nucleus / nucleotide binding / RNA binding / metal ion binding
Influenza RNA-dependent RNA polymerase subunit PA / RdRp of negative ssRNA viruses with segmented genomes catalytic domain profile. / Influenza RNA-dependent RNA polymerase subunit PB2 / Influenza RNA-dependent RNA polymerase subunit PA / Influenza RNA-dependent RNA polymerase subunit PB1 / 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
RNA-directed RNA polymerase catalytic subunit / Polymerase acidic protein / Polymerase basic protein 2
Biological speciesInfluenza B virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsCusack, S. / Kouba, T.
CitationJournal: Nat. Struct. Mol. Biol. / Year: 2019
Title: Structural snapshots of actively transcribing influenza polymerase.
Authors: Tomas Kouba / Petra Drncová / Stephen Cusack /
Abstract: Influenza virus RNA-dependent RNA polymerase uses unique mechanisms to transcribe its single-stranded genomic viral RNA (vRNA) into messenger RNA. The polymerase is initially bound to a promoter ...Influenza virus RNA-dependent RNA polymerase uses unique mechanisms to transcribe its single-stranded genomic viral RNA (vRNA) into messenger RNA. The polymerase is initially bound to a promoter comprising the partially base-paired 3' and 5' extremities of the RNA. A short, capped primer, 'cap-snatched' from a nascent host polymerase II transcript, is directed towards the polymerase active site to initiate RNA synthesis. Here we present structural snapshots, as determined by X-ray crystallography and cryo-electron microscopy, of actively initiating influenza polymerase as it transitions towards processive elongation. Unexpected conformational changes unblock the active site cavity to allow establishment of a nine-base-pair template-product RNA duplex before the strands separate into distinct exit channels. Concomitantly, as the template translocates, the promoter base pairs are broken and the template entry region is remodeled. These structures reveal details of the influenza polymerase active site that will help optimize nucleoside analogs or other compounds that directly inhibit viral RNA synthesis.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Dec 30, 2018 / Release: Jun 5, 2019
RevisionDateData content typeGroupCategoryItemProviderType
1.0Jun 5, 2019Structure modelrepositoryInitial release
1.1Jun 12, 2019Structure modelData collection / Database referencescitation / citation_author / database_PDB_rev / database_PDB_rev_record / em_admin / pdbx_database_proc_citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _em_admin.last_update
1.2Jun 19, 2019Structure modelData collection / Database referencescitation / citation_author / em_admin / pdbx_database_proc_citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update

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Assembly

Deposited unit
A: Polymerase acidic protein
B: RNA-directed RNA polymerase catalytic subunit
C: Polymerase basic protein 2
M: Capped RNA
V: 5 end
R: 3 end
hetero molecules


Theoretical massNumber of molelcules
Total (without water)279,2478
Polymers279,1996
Non-polymers492
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA

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Components

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Protein/peptide , 3 types, 3 molecules ABC

#1: Protein/peptide 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, EC: 3.1.-.-
#2: Protein/peptide 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, EC: 2.7.7.48
#3: Protein/peptide Polymerase basic protein 2 / RNA-directed RNA polymerase subunit P3


Mass: 90844.109 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: U3RSD4

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RNA chain , 3 types, 3 molecules MVR

#4: RNA chain Capped RNA


Mass: 5241.141 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Influenza B virus
#5: RNA chain 5 end


Mass: 4557.820 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Influenza B virus
#6: RNA chain 3 end


Mass: 6525.820 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Influenza B virus

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Non-polymers , 1 types, 2 molecules

#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Magnesium

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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

Name: Influenza polymerase pre-initiation complex / Type: COMPLEX

IDEntity IDParent-IDSource
11, 2, 3, 4, 5, 60MULTIPLE SOURCES
21, 2, 31RECOMBINANT
34, 5, 61RECOMBINANT
Source (natural)

Ncbi tax-ID: 11520 / Organism: Influenza B virus

IDEntity assembly-ID
22
33
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Trichoplusia ni (cabbage looper)7111
33synthetic construct (others)32630
Buffer solutionpH: 7.4
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: 22 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 30000 / Symmetry type: POINT

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