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- PDB-7qtl: Influenza A/H7N9 polymerase elongation complex -

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Basic information

Entry
Database: PDB / ID: 7qtl
TitleInfluenza A/H7N9 polymerase elongation complex
Components
  • 3' vRNA
  • 5' vRNA
  • Polymerase acidic protein
  • Polymerase basic protein 2
  • RNA-directed RNA polymerase catalytic subunit
  • mRNA
KeywordsVIRAL PROTEIN / Influenza / viral RNA-dependent RNA polymerase / cap-dependent transcription
Function / homology
Function and homology information


cap snatching / viral transcription / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / 7-methylguanosine mRNA capping / host cell mitochondrion / virion component / endonuclease activity / Hydrolases; Acting on ester bonds / host cell cytoplasm / symbiont-mediated suppression of host gene expression ...cap snatching / viral transcription / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / 7-methylguanosine mRNA capping / host cell mitochondrion / virion component / endonuclease activity / Hydrolases; Acting on ester bonds / host cell cytoplasm / symbiont-mediated suppression of host gene expression / viral translational frameshifting / RNA-directed RNA polymerase / viral RNA genome replication / nucleotide binding / RNA-directed RNA polymerase activity / DNA-templated transcription / host cell nucleus / RNA binding / metal ion binding
Similarity search - Function
Influenza RNA-dependent RNA polymerase subunit PA, endonuclease domain / Restriction Endonuclease / Influenza RNA-dependent RNA polymerase subunit PB2 / 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 / Influenza RNA-dependent RNA polymerase subunit PB2 / 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 / Polymerase acidic protein / 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
Chem-2TM / Chem-GTA / RNA / RNA (> 10) / Polymerase acidic protein / RNA-directed RNA polymerase catalytic subunit / Polymerase basic protein 2
Similarity search - Component
Biological speciesInfluenza A virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.48 Å
AuthorsCusack, S. / Kouba, T.
Funding support France, 1items
OrganizationGrant numberCountry
French National Research Agency France
CitationJournal: Cell Rep / Year: 2023
Title: Direct observation of backtracking by influenza A and B polymerases upon consecutive incorporation of the nucleoside analog T1106.
Authors: Tomas Kouba / Anna Dubankova / Petra Drncova / Elisa Donati / Pietro Vidossich / Valentina Speranzini / Alex Pflug / Johanna Huchting / Chris Meier / Marco De Vivo / Stephen Cusack /
Abstract: The antiviral pseudo-base T705 and its de-fluoro analog T1106 mimic adenine or guanine and can be competitively incorporated into nascent RNA by viral RNA-dependent RNA polymerases. Although ...The antiviral pseudo-base T705 and its de-fluoro analog T1106 mimic adenine or guanine and can be competitively incorporated into nascent RNA by viral RNA-dependent RNA polymerases. Although dispersed, single pseudo-base incorporation is mutagenic, consecutive incorporation causes polymerase stalling and chain termination. Using a template encoding single and then consecutive T1106 incorporation four nucleotides later, we obtained a cryogenic electron microscopy structure of stalled influenza A/H7N9 polymerase. This shows that the entire product-template duplex backtracks by 5 nt, bringing the singly incorporated T1106 to the +1 position, where it forms an unexpected T1106:U wobble base pair. Similar structures show that influenza B polymerase also backtracks after consecutive T1106 incorporation, regardless of whether prior single incorporation has occurred. These results give insight into the unusual mechanism of chain termination by pyrazinecarboxamide base analogs. Consecutive incorporation destabilizes the proximal end of the product-template duplex, promoting irreversible backtracking to a more energetically favorable overall configuration.
History
DepositionJan 14, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 28, 2022Provider: repository / Type: Initial release
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Revision 1.1Jan 18, 2023Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)273,29410
Polymers271,9776
Non-polymers1,3174
Water2,162120
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area46560 Å2
ΔGint-299 kcal/mol
Surface area84530 Å2

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Components

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Protein , 3 types, 3 molecules ABC

#1: Protein Polymerase acidic protein / RNA-directed RNA polymerase subunit P2


Mass: 82930.602 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (A/Zhejiang/DTID-ZJU01/2013(H7N9))
Strain: A/Zhejiang/DTID-ZJU01/2013(H7N9) / Gene: PA / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: M9TI86, 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: 86496.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (A/Zhejiang/DTID-ZJU01/2013(H7N9))
Strain: A/Zhejiang/DTID-ZJU01/2013(H7N9) / Gene: PB1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: M9TLW3, RNA-directed RNA polymerase
#3: Protein Polymerase basic protein 2 / RNA-directed RNA polymerase subunit P3


Mass: 86007.320 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (A/Zhejiang/DTID-ZJU01/2013(H7N9))
Strain: A/Zhejiang/DTID-ZJU01/2013(H7N9) / Gene: PB2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: X5F427

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RNA chain , 3 types, 3 molecules VRM

#4: RNA chain 5' vRNA


Mass: 4557.820 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Influenza A virus
#5: RNA chain 3' vRNA


Mass: 6525.820 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Influenza A virus
#6: RNA chain mRNA


Mass: 5459.334 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Influenza A virus

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Non-polymers , 4 types, 124 molecules

#7: Chemical ChemComp-2TM / 5'-O-[(S)-hydroxy{[(S)-hydroxy(phosphonooxy)phosphoryl]methyl}phosphoryl]cytidine / CMPcPP


Mass: 481.184 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H18N3O13P3 / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#9: Chemical ChemComp-GTA / P1-7-METHYLGUANOSINE-P3-ADENOSINE-5',5'-TRIPHOSPHATE / 7-METHYL-GPPPA


Mass: 787.441 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H30N10O17P3
#10: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 120 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationN

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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: A/H7N9-Elongation / Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT
Molecular weightValue: 0.255 MDa / Experimental value: YES
Source (natural)Organism: Influenza A virus (A/Zhejiang/DTID-ZJU01/2013(H7N9))
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
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 FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 33.4 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.48 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 116539 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00318719
ELECTRON MICROSCOPYf_angle_d0.6125467
ELECTRON MICROSCOPYf_dihedral_angle_d10.1782956
ELECTRON MICROSCOPYf_chiral_restr0.0412846
ELECTRON MICROSCOPYf_plane_restr0.0083108

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