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- PDB-6qx3: Influenza A virus (A/NT/60/1968) polymerase Hetermotrimer in comp... -

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Basic information

Entry
Database: PDB / ID: 6qx3
TitleInfluenza A virus (A/NT/60/1968) polymerase Hetermotrimer in complex with 3'5' cRNA promoter and Nb8205
Components
  • Nb8205
  • Polymerase acidic protein
  • Polymerase basic protein 2
  • RNA (5'-R(P*AP*GP*CP*AP*AP*AP*AP*GP*CP*A)-3')
  • RNA (5'-R(P*UP*UP*CP*U)-3')
  • RNA-directed RNA polymerase catalytic subunit
KeywordsRNA BINDING PROTEIN / Influenza A / RNA polymerase / Influenza polymerase / Influenza dimer / RDRP
Function / homology
Function and homology information


cap snatching / suppression by virus of host RNA polymerase II activity / host cell mitochondrion / 7-methylguanosine mRNA capping / viral transcription / suppression by virus of host MAVS activity / virion / RNA-directed RNA polymerase / viral RNA genome replication / endonuclease activity ...cap snatching / suppression by virus of host RNA polymerase II activity / host cell mitochondrion / 7-methylguanosine mRNA capping / viral transcription / suppression by virus of host MAVS activity / virion / RNA-directed RNA polymerase / viral RNA genome replication / endonuclease activity / RNA-directed 5'-3' RNA polymerase activity / host cell cytoplasm / Hydrolases, Acting on ester bonds / transcription, DNA-templated / host cell nucleus / nucleotide binding / RNA binding / metal ion binding
Influenza RNA-dependent RNA polymerase subunit PB2 / RdRp of negative ssRNA viruses with segmented genomes catalytic domain profile. / Influenza RNA-dependent RNA polymerase subunit PA / Influenza RNA-dependent RNA polymerase subunit PB1 / PA/PA-X superfamily / Polymerase acidic protein / PB2, C-terminal / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB2 / Influenza RNA-dependent RNA polymerase subunit PA / RNA-directed RNA polymerase, negative-strand RNA virus
Polymerase basic protein 2 / Polymerase acidic protein / RNA-directed RNA polymerase catalytic subunit
Biological speciesLama glama (llama)
Influenza A virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.79 Å
AuthorsCarrique, L. / Keown, J.R. / Fan, H. / Fodor, E. / Grimes, J.M.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Wellcome Trust200835/Z/16/Z United Kingdom
Medical Research Council (United Kingdom)MR/R009945/1 United Kingdom
Medical Research Council (United Kingdom)MR/K000241/1 United Kingdom
CitationJournal: Nature / Year: 2019
Title: Structures of influenza A virus RNA polymerase offer insight into viral genome replication.
Authors: Haitian Fan / Alexander P Walker / Loïc Carrique / Jeremy R Keown / Itziar Serna Martin / Dimple Karia / Jane Sharps / Narin Hengrung / Els Pardon / Jan Steyaert / Jonathan M Grimes / Ervin Fodor /
Abstract: Influenza A viruses are responsible for seasonal epidemics, and pandemics can arise from the transmission of novel zoonotic influenza A viruses to humans. Influenza A viruses contain a segmented ...Influenza A viruses are responsible for seasonal epidemics, and pandemics can arise from the transmission of novel zoonotic influenza A viruses to humans. Influenza A viruses contain a segmented negative-sense RNA genome, which is transcribed and replicated by the viral-RNA-dependent RNA polymerase (FluPol) composed of PB1, PB2 and PA subunits. Although the high-resolution crystal structure of FluPol of bat influenza A virus has previously been reported, there are no complete structures available for human and avian FluPol. Furthermore, the molecular mechanisms of genomic viral RNA (vRNA) replication-which proceeds through a complementary RNA (cRNA) replicative intermediate, and requires oligomerization of the polymerase-remain largely unknown. Here, using crystallography and cryo-electron microscopy, we determine the structures of FluPol from human influenza A/NT/60/1968 (H3N2) and avian influenza A/duck/Fujian/01/2002 (H5N1) viruses at a resolution of 3.0-4.3 Å, in the presence or absence of a cRNA or vRNA template. In solution, FluPol forms dimers of heterotrimers through the C-terminal domain of the PA subunit, the thumb subdomain of PB1 and the N1 subdomain of PB2. The cryo-electron microscopy structure of monomeric FluPol bound to the cRNA template reveals a binding site for the 3' cRNA at the dimer interface. We use a combination of cell-based and in vitro assays to show that the interface of the FluPol dimer is required for vRNA synthesis during replication of the viral genome. We also show that a nanobody (a single-domain antibody) that interferes with FluPol dimerization inhibits the synthesis of vRNA and, consequently, inhibits virus replication in infected cells. Our study provides high-resolution structures of medically relevant FluPol, as well as insights into the replication mechanisms of the viral RNA genome. In addition, our work identifies sites in FluPol that could be targeted in the development of antiviral drugs.
Validation Report
SummaryFull reportAbout validation report
History
DepositionMar 7, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 4, 2019Provider: repository / Type: Initial release
Revision 1.1Sep 11, 2019Group: Data collection / Other / Category: cell
Item: _cell.length_a / _cell.length_b ..._cell.length_a / _cell.length_b / _cell.length_c / _cell.volume
Revision 1.2Sep 18, 2019Group: Data collection / Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

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Assembly

Deposited unit
D: RNA (5'-R(P*AP*GP*CP*AP*AP*AP*AP*GP*CP*A)-3')
G: RNA (5'-R(P*UP*UP*CP*U)-3')
O: Nb8205
B: RNA-directed RNA polymerase catalytic subunit
A: Polymerase acidic protein
C: Polymerase basic protein 2


Theoretical massNumber of molelcules
Total (without water)280,7056
Polymers280,7056
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area27290 Å2
ΔGint-157 kcal/mol
Surface area55920 Å2
MethodPISA

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Components

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RNA chain , 2 types, 2 molecules DG

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


Mass: 4901.097 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Influenza A virus (A/nt/60/1968(H3N2))
#2: RNA chain RNA (5'-R(P*UP*UP*CP*U)-3')


Mass: 4683.753 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Influenza A virus (A/nt/60/1968(H3N2))

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Protein/peptide , 4 types, 4 molecules OBAC

#3: Protein/peptide Nb8205


Mass: 14835.375 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli (E. coli)
#4: Protein/peptide RNA-directed RNA polymerase catalytic subunit / Polymerase basic protein 1 / PB1 / RNA-directed RNA polymerase subunit P1


Mass: 86524.086 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (strain A/Hong Kong/1/1968 H3N2)
Strain: A/Hong Kong/1/1968 H3N2 / Gene: PB1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q910D6, RNA-directed RNA polymerase
#5: Protein/peptide Polymerase acidic protein / RNA-directed RNA polymerase subunit P2


Mass: 83100.797 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (strain A/Northern Territory/60/1968 H3N2)
Strain: A/Northern Territory/60/1968 H3N2 / Gene: PA / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P03434, Hydrolases, Acting on ester bonds
#6: Protein/peptide Polymerase basic protein 2 / RNA-directed RNA polymerase subunit P3


Mass: 86659.898 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (strain A/Northern Territory/60/1968 H3N2)
Strain: A/Northern Territory/60/1968 H3N2 / Gene: PB2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P03429

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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: Influenza A virus (A/NT/60/1968) polymerase Hetermotrimer in complex with 3'5' cRNA promoter and Nb8205
Type: COMPLEX
Details: Sample was treated with 0.001% glutaraldehyde for 20 min on ice prior quenching with 100 mM Tris-HCl pH 7.5 and gel filtration.
Entity ID: 1, 2, 3, 4, 5, 6 / Source: RECOMBINANT
Molecular weightValue: 0.25 MDa / Experimental value: NO
Source (natural)Organism: Influenza A virus (A/nt/60/1968(H3N2))
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Details: Sample was purified in 20 mM HEPES, pH 7.5, 150 mM NaCl with Tween 20 added to a final concentration 0f 0.05% prior to plunging grids.
Buffer component

Buffer-ID: 1

IDConc.NameFormula
120 mMHEPES
2150 mMNaClSodium chloride
SpecimenConc.: 0.35 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: C-flat
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K / Details: blot for 3.5 sec before plunging

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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 / Nominal magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1300 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6 sec. / Electron dose: 1.25 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2456
EM imaging opticsPhase plate: Volta phase plate
Image scansMovie frames/image: 24

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.14_3260refinement
PHENIX1.14_3260refinement
EM software
IDNameVersionCategory
1cryoSPARC2.5particle selection
2EPUimage acquisition
4Gctf1.18CTF correction
7UCSF Chimera1.13model fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
19PHENIX1.14model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 505860 / Details: template picking in cryosparc v2.5
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.79 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 52932 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 110 / Protocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6QPG
RefinementStereochemistry target values: CDL v1.2
Refine LS restraints

Refinement-ID: ELECTRON MICROSCOPY

TypeDev idealNumber
f_bond_d0.00521852
f_angle_d0.733839435
f_chiral_restr0.0431666
f_plane_restr0.00333190
f_dihedral_angle_d8.47698799

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