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- PDB-7nir: 1918 H1N1 Viral influenza polymerase heterotrimer with Nb8191 core -

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Basic information

Entry
Database: PDB / ID: 7nir
Title1918 H1N1 Viral influenza polymerase heterotrimer with Nb8191 core
Components
  • Nanobody8191 core
  • 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 / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / cap snatching / 7-methylguanosine mRNA capping / viral transcription / host cell mitochondrion / 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 / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / cap snatching / 7-methylguanosine mRNA capping / viral transcription / host cell mitochondrion / 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 RNA genome replication / RNA-dependent RNA polymerase activity / 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 / Lysin motif / Influenza RNA-dependent RNA polymerase subunit PB1 / LysM domain superfamily / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB2 / PB2, C-terminal ...Octapeptide repeat / Octapeptide repeat / Protein A, Ig-binding domain / B domain / Lysin motif / Influenza RNA-dependent RNA polymerase subunit PB1 / LysM domain superfamily / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB2 / PB2, C-terminal / LysM domain / LysM domain profile. / : / : / : / : / : / Influenza RNA polymerase PB2 N-terminal region / Influenza RNA polymerase PB2 second domain / Influenza RNA polymerase PB2 middle domain / Influenza RNA polymerase PB2 6th domain / Influenza RNA polymerase PB2 C-terminal domain / : / : / Influenza RNA polymerase PB2 helical domain / LysM domain / Influenza RNA polymerase PB2 CAP binding domain / Immunoglobulin/albumin-binding domain superfamily / Polymerase acidic protein / YSIRK type signal peptide / 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. / YSIRK Gram-positive signal peptide / 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: 6.7 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 13, 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-12363
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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: Nanobody8191 core


Theoretical massNumber of molelcules
Total (without water)296,3576
Polymers296,3576
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area39390 Å2
ΔGint-238 kcal/mol
Surface area70350 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 (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

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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 Nanobody8191 core


Mass: 14449.850 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 Nb8191COMPLEXall0MULTIPLE SOURCES
2Polymerase acidic protein, RNA-directed RNA polymerase catalytic subunit, Polymerase basic protein 2COMPLEX#1-#31RECOMBINANT
3RNACOMPLEX#4-#51RECOMBINANT
4Nanobody8191 coreCOMPLEX#61RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Influenza A virus (strain A/Brevig Mission/1/1918 H1N1)88776
23Influenza A virus (strain 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 chlorideNaClSodium chloride1
225 mMSodium thiocynateNaSCN1
320 mMHepesHEPES-Na1
40.005 %Tween-20Tween-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

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: 51.8 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.1_4122refinement
PHENIX1.19.1_4122refinement
EM software
IDNameVersionCategory
4cryoSPARC2.15CTF correction
7UCSF Chimera1.19model fitting
9cryoSPARC2.15initial Euler assignment
12cryoSPARC2.153D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 6.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 33278 / 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_d0.004315273
ELECTRON MICROSCOPYf_angle_d0.689420702
ELECTRON MICROSCOPYf_chiral_restr0.04362276
ELECTRON MICROSCOPYf_plane_restr0.0052585
ELECTRON MICROSCOPYf_dihedral_angle_d13.46725898

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