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- PDB-6xzd: Influenza C virus polymerase complex without chicken ANP32A - Sub... -

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Basic information

Entry
Database: PDB / ID: 6xzd
TitleInfluenza C virus polymerase complex without chicken ANP32A - Subclass 2
Components
  • Polymerase acidic protein
  • Polymerase basic protein 2
  • RNA (5'-R(*AP*GP*UP*AP*GP*AP*AP*AP*CP*AP*AP*GP*GP*GP*CP*CP*CP*UP*GP*C)-3')
  • RNA-directed RNA polymerase catalytic subunit
KeywordsVIRAL PROTEIN / Influenza Polymerase / ANP32 / replication / RNA-dependent RNA polymerase
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 / virion component / endonuclease activity / host cell cytoplasm / Hydrolases; Acting on ester bonds / RNA-directed RNA polymerase / viral translational frameshifting ...cap snatching / viral transcription / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / 7-methylguanosine mRNA capping / 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 / nucleotide binding / DNA-templated transcription / host cell nucleus / RNA binding / metal ion binding
Similarity search - Function
Influenza RNA-dependent RNA polymerase subunit PB2 / 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-dependent RNA polymerase subunit PB2 / 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.
Similarity search - Domain/homology
RNA / RNA (> 10) / Polymerase basic protein 2 / RNA-directed RNA polymerase catalytic subunit / Polymerase acidic protein
Similarity search - Component
Biological speciesInfluenza C virus
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsKeown, J.R. / Carrique, L. / Fan, H. / Grimes, J.M. / Fodor, E.
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: Nature / Year: 2020
Title: Host ANP32A mediates the assembly of the influenza virus replicase.
Authors: Loïc Carrique / Haitian Fan / Alexander P Walker / Jeremy R Keown / Jane Sharps / Ecco Staller / Wendy S Barclay / Ervin Fodor / Jonathan M Grimes /
Abstract: Aquatic birds represent a vast reservoir from which new pandemic influenza A viruses can emerge. Influenza viruses contain a negative-sense segmented RNA genome that is transcribed and replicated by ...Aquatic birds represent a vast reservoir from which new pandemic influenza A viruses can emerge. Influenza viruses contain a negative-sense segmented RNA genome that is transcribed and replicated by the viral heterotrimeric RNA polymerase (FluPol) in the context of viral ribonucleoprotein complexes. RNA polymerases of avian influenza A viruses (FluPolA) replicate viral RNA inefficiently in human cells because of species-specific differences in acidic nuclear phosphoprotein 32 (ANP32), a family of essential host proteins for FluPol activity. Host-adaptive mutations, particularly a glutamic-acid-to-lysine mutation at amino acid residue 627 (E627K) in the 627 domain of the PB2 subunit, enable avian FluPolA to overcome this restriction and efficiently replicate viral RNA in the presence of human ANP32 proteins. However, the molecular mechanisms of genome replication and the interplay with ANP32 proteins remain largely unknown. Here we report cryo-electron microscopy structures of influenza C virus polymerase (FluPolC) in complex with human and chicken ANP32A. In both structures, two FluPolC molecules form an asymmetric dimer bridged by the N-terminal leucine-rich repeat domain of ANP32A. The C-terminal low-complexity acidic region of ANP32A inserts between the two juxtaposed PB2 627 domains of the asymmetric FluPolA dimer, suggesting a mechanism for how the adaptive PB2(E627K) mutation enables the replication of viral RNA in mammalian hosts. We propose that this complex represents a replication platform for the viral RNA genome, in which one of the FluPol molecules acts as a replicase while the other initiates the assembly of the nascent replication product into a viral ribonucleoprotein complex.
History
DepositionFeb 4, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 23, 2020Provider: repository / Type: Initial release
Revision 1.1May 22, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
IN1: RNA (5'-R(*AP*GP*UP*AP*GP*AP*AP*AP*CP*AP*AP*GP*GP*GP*CP*CP*CP*UP*GP*C)-3')
AP1: Polymerase acidic protein
BP1: RNA-directed RNA polymerase catalytic subunit
CP1: Polymerase basic protein 2
DP1: Polymerase acidic protein
EP1: RNA-directed RNA polymerase catalytic subunit
FP1: Polymerase basic protein 2


Theoretical massNumber of molelcules
Total (without water)527,1447
Polymers527,1447
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area65790 Å2
ΔGint-375 kcal/mol
Surface area161320 Å2
MethodPISA

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Components

#1: RNA chain RNA (5'-R(*AP*GP*UP*AP*GP*AP*AP*AP*CP*AP*AP*GP*GP*GP*CP*CP*CP*UP*GP*C)-3')


Mass: 14901.737 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: Protein Polymerase acidic protein / RNA-directed RNA polymerase subunit P2


Mass: 82025.531 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza C virus (strain C/Johannesburg/1/1966)
Strain: C/Johannesburg/1/1966 / Gene: PA, P3 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: Q9IMP5, Hydrolases; Acting on ester bonds
#3: Protein RNA-directed RNA polymerase catalytic subunit / Polymerase basic protein 1 / PB1 / RNA-directed RNA polymerase subunit P1


Mass: 86145.945 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza C virus (strain C/Johannesburg/1/1966)
Strain: C/Johannesburg/1/1966 / Gene: PB1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9IMP4, RNA-directed RNA polymerase
#4: Protein Polymerase basic protein 2 / RNA-directed RNA polymerase subunit P3


Mass: 87949.422 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza C virus (strain C/Johannesburg/1/1966)
Strain: C/Johannesburg/1/1966 / Gene: PB2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9IMP3

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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-IDSourceDetails
1Influenza C virus polymerase in complex with chicken ANP32ACOMPLEXall0MULTIPLE SOURCES
2Influenza C virus polymerasesCOMPLEX#2-#41RECOMBINANT
4Influenza viral RNA (vRNA) promoter 47merCOMPLEX#11RECOMBINANTSynthetic RNA
Molecular weight
IDEntity assembly-IDExperimental value
11NO
22
34
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Influenza C virus (C/Johannesburg/1/66)100673
32Influenza C virus (C/Johannesburg/1/66)100673
54Synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Spodoptera frugiperda (fall armyworm)7108
32Spodoptera frugiperda (fall armyworm)7108
54Synthetic construct (others)32630
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaCL1
30.005 %Tween 20C58H114O261
SpecimenConc.: 0.28 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 294 K

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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 magnification: 105000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 2000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 11 sec. / Electron dose: 38.8 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3678
Details: Single specimen tilt of 30 degrees for around two third of the images
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 44 / Used frames/image: 1-44

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18rc2_3794refinement
PHENIX1.18rc2_3794refinement
EM software
IDNameVersionCategory
2EPU2.1image acquisition
4cryoSPARC2.12CTF correction
7Coot0.9model fitting
9cryoSPARC2.12initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
13PHENIX1.17model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2534332
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 169000 / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 55.76 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.012432437
ELECTRON MICROSCOPYf_angle_d0.903543725
ELECTRON MICROSCOPYf_chiral_restr0.05964821
ELECTRON MICROSCOPYf_plane_restr0.00545520
ELECTRON MICROSCOPYf_dihedral_angle_d16.29712529

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