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- EMDB-4664: Influenza A virus (A/NT/60/1968) polymerase dimer of Hetermotrime... -

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Entry
Database: EMDB / ID: EMD-4664
TitleInfluenza A virus (A/NT/60/1968) polymerase dimer of Hetermotrimer in complex with 3'5' cRNA promoter
Map data
SampleInfluenza A virus (A/NT/60/1968) polymerase dimer of heterotrimer in complex with 5' cRNA promoter
Biological speciesInfluenza A virus (A/nt/60/1968(H3N2))
Methodsingle particle reconstruction / cryo EM / Resolution: 4.34 Å
AuthorsCarrique L / Keown JR
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.
History
DepositionMar 7, 2019-
Header (metadata) releaseMar 20, 2019-
UpdateMar 20, 2019-
Current statusMar 20, 2019Processing site: PDBe / Status: On hold (released when the article is published)

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Structure visualization

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Map

FileOn hold (released when the article is published)
Voxel sizeX=Y=Z: 1.043 Å
Density
Contour LevelBy AUTHOR: 0.0205
Minimum - Maximum-0.077777736 - 0.12670113
Average (Standard dev.)0.00005305033 (±0.0037717356)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions250250250
Spacing250250250
CellA=B=C: 260.75 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire Influenza A virus (A/NT/60/1968) polymerase dimer of heterotrimer...

EntireName: Influenza A virus (A/NT/60/1968) polymerase dimer of heterotrimer in complex with 5' cRNA promoter
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.
Number of components: 1

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Component #1: protein, Influenza A virus (A/NT/60/1968) polymerase dimer of het...

ProteinName: Influenza A virus (A/NT/60/1968) polymerase dimer of heterotrimer in complex with 5' cRNA promoter
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.
Recombinant expression: No
MassTheoretical: 250 kDa
SourceSpecies: Influenza A virus (A/nt/60/1968(H3N2))
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.35 mg/mL
Buffer solution: 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.
pH: 7.5
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 298 K / Humidity: 100 % / Details: blot for 3.5 sec before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.32 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 130000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 500.0 - 700.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 1178

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 36913
3D reconstructionSoftware: RELION / Resolution: 4.34 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Refinement space: REAL / Overall bvalue: 80

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