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- PDB-7nk2: 1918 H1N1 Viral influenza polymerase heterotrimer with Nb8202 core -

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Basic information

Entry
Database: PDB / ID: 7nk2
Title1918 H1N1 Viral influenza polymerase heterotrimer with Nb8202 core
Components
  • Nanobody8202
  • 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 / nanobody
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)
Camelidae mixed library (mammal)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.84 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 17, 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

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Structure visualization

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  • Deposited structure unit
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  • EMDB-12429
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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')
F: Nanobody8202


Theoretical massNumber of molelcules
Total (without water)296,8346
Polymers296,8346
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area38800 Å2
ΔGint-237 kcal/mol
Surface area70960 Å2
MethodPISA

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Components

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Protein , 2 types, 2 molecules AB

#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 (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 (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

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RNA chain , 2 types, 2 molecules ED

#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
Source: (synth.) Influenza A virus (A/Brevig Mission/1/1918(H1N1))
#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
Source: (synth.) Influenza A virus (A/Brevig Mission/1/1918(H1N1))

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Antibody , 2 types, 2 molecules CF

#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
#6: Antibody Nanobody8202


Mass: 14926.537 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Camelidae mixed library (mammal) / Production host: Escherichia coli (E. coli)

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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 promoters and Nb8202COMPLEXall0MULTIPLE SOURCES
2RNA-directed RNA polymerase catalytic subunit, Polymerase acidic protein, Polymerase basic protein 2COMPLEX#1-#31RECOMBINANT
3RNACOMPLEX#4-#51RECOMBINANT
4Nb8202 coreCOMPLEX#61RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Influenza A virus (A/Brevig Mission/1/1918(H1N1))88776
23Influenza A virus (A/Brevig Mission/1/1918(H1N1))88776
34Camelidae mixed library (mammal)1579311
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Spodoptera frugiperda (fall armyworm)7108
23synthetic construct (others)32630
34Escherichia coli (E. coli)562
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1166 mMsodium chlorideNaCl1
225 mMsodium thiocynateNaSCN1
320 mMHepesHEPES-Na1
40.005 %Tween20Tween-201
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 69.5 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.19.1_4122refinement
PHENIX1.19.1_4122refinement
EM software
IDNameVersionCategory
7UCSF Chimera1.19model fitting
10cryoSPARC2.15initial Euler assignment
11cryoSPARC2.15final Euler assignment
12cryoSPARC2.15classification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1277000
3D reconstructionResolution: 4.84 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 198338 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 7HNA

7hna

RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 30 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004415256
ELECTRON MICROSCOPYf_angle_d0.730120676
ELECTRON MICROSCOPYf_chiral_restr0.04652266
ELECTRON MICROSCOPYf_plane_restr0.00512583
ELECTRON MICROSCOPYf_dihedral_angle_d13.60985889

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