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- PDB-8r1l: Structure of avian H5N1 influenza A polymerase in complex with hu... -

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Basic information

Entry
Database: PDB / ID: 8r1l
TitleStructure of avian H5N1 influenza A polymerase in complex with human ANP32B.
Components
  • Acidic leucine-rich nuclear phosphoprotein 32 family member B
  • Polymerase acidic protein
  • Polymerase basic protein 2
  • RNA-directed RNA polymerase catalytic subunit
KeywordsVIRAL PROTEIN / Influenza virus / replication platform / H5N1 / influenza adaptive mutations / host adaptation / viral polymerase / cryo-EM / HPAI.
Function / homology
Function and homology information


ventricular system development / vasculature development / RNA polymerase binding / roof of mouth development / cap snatching / viral transcription / inner ear development / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / negative regulation of cell differentiation / positive regulation of protein export from nucleus ...ventricular system development / vasculature development / RNA polymerase binding / roof of mouth development / cap snatching / viral transcription / inner ear development / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / negative regulation of cell differentiation / positive regulation of protein export from nucleus / activation of cysteine-type endopeptidase activity involved in apoptotic process / nucleosome assembly / histone binding / endonuclease activity / regulation of apoptotic process / host cell cytoplasm / RNA-directed RNA polymerase / viral RNA genome replication / nucleotide binding / RNA-dependent RNA polymerase activity / host cell nucleus / RNA binding / extracellular exosome / nucleoplasm / metal ion binding / nucleus / cytoplasm
Similarity search - Function
Acidic leucine-rich nuclear phosphoprotein 32 / U2A'/phosphoprotein 32 family A, C-terminal / occurring C-terminal to leucine-rich repeats / Leucine-rich repeat / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB1 / 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 ...Acidic leucine-rich nuclear phosphoprotein 32 / U2A'/phosphoprotein 32 family A, C-terminal / occurring C-terminal to leucine-rich repeats / Leucine-rich repeat / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB1 / 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. / Leucine-rich repeat profile. / Leucine-rich repeat / Leucine-rich repeat domain superfamily
Similarity search - Domain/homology
RNA-directed RNA polymerase catalytic subunit / Polymerase acidic protein / Acidic leucine-rich nuclear phosphoprotein 32 family member B
Similarity search - Component
Biological speciesHomo sapiens (human)
Influenza A virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsCarrique, L. / Staller, E. / Keown, J.R. / Fan, H. / Fodor, E. / Grimes, J.M.
Funding support United Kingdom, 6items
OrganizationGrant numberCountry
Wellcome Trust200835/Z/16/Z United Kingdom
Wellcome Trust222510/Z/21/Z United Kingdom
Medical Research Council (MRC, United Kingdom)MR/R009945/1 United Kingdom
Wellcome Trust093305/Z/10/Z United Kingdom
Wellcome Trust060208/Z/00/Z United Kingdom
Wellcome Trust203141/Z/16/Z United Kingdom
CitationJournal: Nat Commun / Year: 2024
Title: Structures of H5N1 influenza polymerase with ANP32B reveal mechanisms of genome replication and host adaptation.
Authors: Ecco Staller / Loïc Carrique / Olivia C Swann / Haitian Fan / Jeremy R Keown / Carol M Sheppard / Wendy S Barclay / Jonathan M Grimes / Ervin Fodor /
Abstract: Avian influenza A viruses (IAVs) pose a public health threat, as they are capable of triggering pandemics by crossing species barriers. Replication of avian IAVs in mammalian cells is hindered by ...Avian influenza A viruses (IAVs) pose a public health threat, as they are capable of triggering pandemics by crossing species barriers. Replication of avian IAVs in mammalian cells is hindered by species-specific variation in acidic nuclear phosphoprotein 32 (ANP32) proteins, which are essential for viral RNA genome replication. Adaptive mutations enable the IAV RNA polymerase (FluPolA) to surmount this barrier. Here, we present cryo-electron microscopy structures of monomeric and dimeric avian H5N1 FluPolA with human ANP32B. ANP32B interacts with the PA subunit of FluPolA in the monomeric form, at the site used for its docking onto the C-terminal domain of host RNA polymerase II during viral transcription. ANP32B acts as a chaperone, guiding FluPolA towards a ribonucleoprotein-associated FluPolA to form an asymmetric dimer-the replication platform for the viral genome. These findings offer insights into the molecular mechanisms governing IAV genome replication, while enhancing our understanding of the molecular processes underpinning mammalian adaptations in avian-origin FluPolA.
History
DepositionNov 2, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 8, 2024Provider: repository / Type: Initial release
Revision 1.1May 29, 2024Group: 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

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
D: Acidic leucine-rich nuclear phosphoprotein 32 family member B
A: Polymerase acidic protein
B: RNA-directed RNA polymerase catalytic subunit
C: Polymerase basic protein 2


Theoretical massNumber of molelcules
Total (without water)299,4134
Polymers299,4134
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Acidic leucine-rich nuclear phosphoprotein 32 family member B


Mass: 28816.652 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ANP32B / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q92688
#2: Protein Polymerase acidic protein


Mass: 82600.281 Da / Num. of mol.: 1 / Mutation: Q556R
Source method: isolated from a genetically manipulated source
Details: Polymerase Acidic Protein A / Source: (gene. exp.) Influenza A virus / Strain: Influenza A virus (A/turkey/Turkey/1/2005(H5N1)) / Gene: PA / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A5Z236
#3: Protein RNA-directed RNA polymerase catalytic subunit


Mass: 86616.312 Da / Num. of mol.: 1 / Mutation: K577E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus / Strain: Influenza A virus (A/turkey/Turkey/1/2005(H5N1)) / Gene: PB1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A5Z231, RNA-directed RNA polymerase
#4: Protein Polymerase basic protein 2


Mass: 101380.016 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus / Strain: Influenza A virus (A/turkey/Turkey/1/2005(H5N1)) / Gene: PB2 / Production host: Spodoptera frugiperda (fall armyworm)

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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: Avian H5N1 apo influenza A polymerase in complex with human ANP32B.
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Influenza A virus / Strain: A/turkey/Turkey/1/2005(H5N1)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
125 mMHEPES1
2150 mMKCl1
35 %Glycerol1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K

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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 FIELDBright-field microscopy / Nominal defocus max: 24000 nm / Nominal defocus min: 5000 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 recordingElectron dose: 50 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k)
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.2particle selection
2EPU3.14image acquisition
4cryoSPARC4.2CTF correction
7UCSF ChimeraXmodel fitting
9Coot0.9.8.7model refinement
10PHENIX1.2model refinement
11cryoSPARCinitial Euler assignment
12cryoSPARC4.2final Euler assignment
13cryoSPARC4.2classification
14cryoSPARCV4.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 63000 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building

3D fitting-ID: 1

IDPDB-IDDetailsSource nameTypeAccession codeInitial refinement model-ID
1ANP32 modelAlphaFoldin silico model
26RR7

6rr7

PDBexperimental model6RR72
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 64.49 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002611299
ELECTRON MICROSCOPYf_angle_d0.483315250
ELECTRON MICROSCOPYf_chiral_restr0.03691678
ELECTRON MICROSCOPYf_plane_restr0.0041961
ELECTRON MICROSCOPYf_dihedral_angle_d4.35971486

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