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

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Basic information

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 dataNone
Sample
  • Complex: Influenza A virus (A/NT/60/1968) polymerase dimer of heterotrimer in complex with 5' cRNA promoter
Function / homology
Function and homology information


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 ...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 / metal ion binding
Similarity search - Function
Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB2 / PB2, C-terminal / : / : / : / : / : / Influenza RNA polymerase PB2 N-terminal region ...Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB2 / PB2, C-terminal / : / : / : / : / : / 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 / Influenza RNA polymerase PB2 CAP binding 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
Polymerase basic protein 2 / RNA-directed RNA polymerase catalytic subunit / Polymerase acidic protein
Similarity search - Component
Biological speciesInfluenza A virus (A/nt/60/1968(H3N2))
Methodsingle particle reconstruction / cryo EM / Resolution: 4.34 Å
AuthorsCarrique L / Keown JR
Funding support United Kingdom, 3 items
OrganizationGrant numberCountry
Wellcome Trust200835/Z/16/Z United Kingdom
Medical Research Council (United Kingdom)MR/K000241/1 United Kingdom
Medical Research Council (United Kingdom)MR/R009945/1 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.
History
DepositionMar 7, 2019-
Header (metadata) releaseMar 20, 2019-
Map releaseJan 15, 2020-
UpdateSep 30, 2020-
Current statusSep 30, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0205
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0205
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4664.png

Downloads & links

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Map

FileDownload / File: emd_4664.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Voxel sizeX=Y=Z: 1.043 Å
Density
Contour LevelBy AUTHOR: 0.0205 / Movie #1: 0.0205
Minimum - Maximum-0.077777736 - 0.12670113
Average (Standard dev.)0.0000530503 (±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 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0431.0431.043
M x/y/z250250250
origin x/y/z0.0000.0000.000
length x/y/z260.750260.750260.750
α/β/γ90.00090.00090.000
start NX/NY/NZ212211153
NX/NY/NZ80102186
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS250250250
D min/max/mean-0.0780.1270.000

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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
Components
  • Complex: Influenza A virus (A/NT/60/1968) polymerase dimer of heterotrimer in complex with 5' cRNA promoter

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Supramolecule #1: Influenza A virus (A/NT/60/1968) polymerase dimer of heterotrimer...

SupramoleculeName: Influenza A virus (A/NT/60/1968) polymerase dimer of heterotrimer in complex with 5' cRNA promoter
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
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.
Source (natural)Organism: Influenza A virus (A/nt/60/1968(H3N2))
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
Molecular weightTheoretical: 250 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.35 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
20.0 mMHEPES
150.0 mMNaClSodium chloride

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.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 3.5 sec before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 0.7 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 130000
Specialist opticsPhase plate: VOLTA PHASE PLATE
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 1178 / Average exposure time: 5.0 sec. / Average electron dose: 1.32 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 56070
CTF correctionSoftware - Name: Gctf (ver. 1.18)
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio model was generated by cryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3) / Details: RELION 3
Final 3D classificationSoftware - Name: RELION (ver. 3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.34 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3) / Number images used: 36913
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 80

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