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- PDB-7nhc: 1918 H1N1 Viral influenza polymerase heterotrimer - Endonuclease ... -

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Basic information

Entry
Database: PDB / ID: 7nhc
Title1918 H1N1 Viral influenza polymerase heterotrimer - Endonuclease ordered (Class2b)
Components
  • Polymerase acidic protein
  • Polymerase basic protein 2,Immunoglobulin G-binding protein A
  • RNA (5'-R(P*AP*GP*UP*AP*GP*AP*AP*AP*CP*AP*AP*GP*GP*CP*C)-3')
  • RNA (5'-R(P*GP*GP*CP*CP*UP*GP*CP*U)-3')
  • RNA-directed RNA polymerase catalytic subunit
KeywordsVIRAL PROTEIN / Influenza / RNA polymerase / H1N1 / 1918
Function / homology
Function and homology information


IgG binding / 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 / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / virion component / endonuclease activity / host cell cytoplasm ...IgG binding / 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 / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / virion component / endonuclease activity / host cell cytoplasm / Hydrolases; Acting on ester bonds / RNA-directed RNA polymerase / viral translational frameshifting / viral RNA genome replication / RNA-dependent RNA polymerase activity / virus-mediated perturbation of host defense response / nucleotide binding / DNA-templated transcription / host cell nucleus / RNA binding / extracellular region / metal ion binding
Similarity search - Function
Octapeptide repeat / Octapeptide repeat / Protein A, Ig-binding domain / B domain / LysM domain superfamily / Lysin motif / LysM domain / LysM domain profile. / Influenza RNA-dependent RNA polymerase subunit PB2 / LysM domain ...Octapeptide repeat / Octapeptide repeat / Protein A, Ig-binding domain / B domain / LysM domain superfamily / Lysin motif / LysM domain / LysM domain profile. / Influenza RNA-dependent RNA polymerase subunit PB2 / LysM domain / 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 / Immunoglobulin/albumin-binding domain superfamily / : / Influenza RNA polymerase PB2 CAP binding domain / : / Influenza RNA polymerase PB2 helical domain / YSIRK type signal peptide / 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 / YSIRK Gram-positive signal peptide / RNA-directed RNA polymerase, negative-strand RNA virus / RdRp of negative ssRNA viruses with segmented genomes catalytic domain profile. / LPXTG cell wall anchor motif / Gram-positive cocci surface proteins LPxTG motif profile. / LPXTG cell wall anchor domain
Similarity search - Domain/homology
RNA / RNA (> 10) / Immunoglobulin G-binding protein A / Polymerase acidic protein / RNA-directed RNA polymerase catalytic subunit / Polymerase basic protein 2
Similarity search - Component
Biological speciesInfluenza A virus
Staphylococcus aureus (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.87 Å
AuthorsKeown, J.R. / Carrique, L. / Fodor, E. / Grimes, J.M.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Wellcome Trust200835/Z/16/Z United Kingdom
Medical Research Council (MRC, United Kingdom)MR/R009945/1 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/K000241/1 United Kingdom
CitationJournal: Nat Commun / Year: 2022
Title: Mapping inhibitory sites on the RNA polymerase of the 1918 pandemic influenza virus using nanobodies.
Authors: Jeremy R Keown / Zihan Zhu / Loïc Carrique / Haitian Fan / Alexander P Walker / Itziar Serna Martin / Els Pardon / Jan Steyaert / Ervin Fodor / Jonathan M Grimes /
Abstract: Influenza A viruses cause seasonal epidemics and global pandemics, representing a considerable burden to healthcare systems. Central to the replication cycle of influenza viruses is the viral RNA- ...Influenza A viruses cause seasonal epidemics and global pandemics, representing a considerable burden to healthcare systems. Central to the replication cycle of influenza viruses is the viral RNA-dependent RNA polymerase which transcribes and replicates the viral RNA genome. The polymerase undergoes conformational rearrangements and interacts with viral and host proteins to perform these functions. Here we determine the structure of the 1918 influenza virus polymerase in transcriptase and replicase conformations using cryo-electron microscopy (cryo-EM). We then structurally and functionally characterise the binding of single-domain nanobodies to the polymerase of the 1918 pandemic influenza virus. Combining these functional and structural data we identify five sites on the polymerase which are sensitive to inhibition by nanobodies. We propose that the binding of nanobodies at these sites either prevents the polymerase from assuming particular functional conformations or interactions with viral or host factors. The polymerase is highly conserved across the influenza A subtypes, suggesting these sites as effective targets for potential influenza antiviral development.
History
DepositionFeb 10, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 1, 2021Provider: repository / Type: Initial release
Revision 1.1Feb 9, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jul 10, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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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,Immunoglobulin G-binding protein A
E: RNA (5'-R(P*GP*GP*CP*CP*UP*GP*CP*U)-3')
D: RNA (5'-R(P*AP*GP*UP*AP*GP*AP*AP*AP*CP*AP*AP*GP*GP*CP*C)-3')


Theoretical massNumber of molelcules
Total (without water)281,9075
Polymers281,9075
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area37880 Å2
ΔGint-231 kcal/mol
Surface area64350 Å2
MethodPISA

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Components

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


Mass: 82707.391 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (strain A/Brevig Mission/1/1918 H1N1)
Strain: A/Brevig Mission/1/1918 H1N1 / Gene: PA / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: Q3HM39, 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: 86625.211 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (strain A/Brevig Mission/1/1918 H1N1)
Strain: A/Brevig Mission/1/1918 H1N1 / Gene: PB1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q3HM40, RNA-directed RNA polymerase
#3: Antibody Polymerase basic protein 2,Immunoglobulin G-binding protein A / RNA-directed RNA polymerase subunit P3 / IgG-binding protein A / Staphylococcal protein A / SpA


Mass: 102377.219 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (strain A/Brevig Mission/1/1918 H1N1), (gene. exp.) Staphylococcus aureus (bacteria)
Strain: A/Brevig Mission/1/1918 H1N1 / Gene: PB2, spa / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q3HM41, UniProt: P38507
#4: RNA chain RNA (5'-R(P*GP*GP*CP*CP*UP*GP*CP*U)-3')


Mass: 5335.125 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: Influenza A virus (A/Brevig Mission/1/1918(H1N1)) / Source: (synth.) synthetic construct (others)
#5: RNA chain RNA (5'-R(P*AP*GP*UP*AP*GP*AP*AP*AP*CP*AP*AP*GP*GP*CP*C)-3')


Mass: 4862.017 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: Influenza A virus (A/Brevig Mission/1/1918(H1N1)) / Source: (synth.) synthetic construct (others)

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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
11918 Influenza virus polymerase heterotirmer in complex with vRNA promotersCOMPLEXall0MULTIPLE SOURCES
21918 Influenza virus polymeraseCOMPLEX#1-#31RECOMBINANT
3vRNA promotersCOMPLEX#4-#51RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Influenza A virus (strain A/Brevig Mission/1/1918 H1N1)88776
23synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Spodoptera frugiperda (fall armyworm)7108
23synthetic construct (others)32630
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMSodium thiocynateNaSCN1
220 mMHEPESHEPES-Na1
3166 mMSodium ChlorideNaCl1
40.0075 % (v/v)Tween 201
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K / Details: 3.5 second blot and 3 microlitres sample.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 5 sec. / Electron dose: 61.5 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 9413
Image scansWidth: 3838 / Height: 3710

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.1_4122refinement
PHENIX1.19.1_4122refinement
EM software
IDNameVersionCategory
2SerialEM3.7image acquisition
4cryoSPARC2.15CTF correction
10cryoSPARC2.15initial Euler assignment
11cryoSPARC2.15final Euler assignment
12cryoSPARC2.15classification
13cryoSPARC2.153D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2854501
Details: Initial automated blob picking followed by template picking.
3D reconstructionResolution: 2.87 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 16827 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 21.91 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002314364
ELECTRON MICROSCOPYf_angle_d0.487619477
ELECTRON MICROSCOPYf_chiral_restr0.03732141
ELECTRON MICROSCOPYf_plane_restr0.00492426
ELECTRON MICROSCOPYf_dihedral_angle_d6.29352108

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