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- EMDB-4666: Influenza A virus (A/NT/60/1968) polymerase dimer of hetermotrime... -

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Basic information

Entry
Database: EMDB / ID: EMD-4666
TitleInfluenza A virus (A/NT/60/1968) polymerase dimer of hetermotrimer in complex with 3'5' cRNA promoter and Nb8205
Map data
SampleInfluenza A virus (A/NT/60/1968) polymerase dimer of hetermotrimer in complex with 3'5' cRNA promoter and Nb8205
  • Nb8205
  • Polymerase acidic protein
  • RNA-directed RNA polymerase catalytic subunit
  • Polymerase basic protein 2
  • nucleic-acidNucleic acid
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 / RNA-directed RNA polymerase catalytic subunit / Polymerase acidic protein
Biological speciesInfluenza A virus (A/nt/60/1968(H3N2)) / Influenza A virus (A/nt/60/1968(H3N2))
Methodsingle particle reconstruction / cryo EM / Resolution: 4.15 Å
AuthorsCarrique L / Keown JR / Fan H / Fodor E / Grimes JM
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.
Validation ReportPDB-ID: 6qxe

SummaryFull reportAbout validation report
History
DepositionMar 7, 2019-
Header (metadata) releaseMar 20, 2019-
Map releaseSep 4, 2019-
UpdateSep 18, 2019-
Current statusSep 18, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.026
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.026
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6qxe
  • Surface level: 0.026
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_4666.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 250 pix.
= 270. Å
1.08 Å/pix.
x 250 pix.
= 270. Å
1.08 Å/pix.
x 250 pix.
= 270. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.026 / Movie #1: 0.026
Minimum - Maximum-0.10696513 - 0.1777088
Average (Standard dev.)0.000054944012 (±0.004839128)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions250250250
Spacing250250250
CellA=B=C: 270.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z250250250
origin x/y/z0.0000.0000.000
length x/y/z270.000270.000270.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-39-149-104
NX/NY/NZ201201211
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS250250250
D min/max/mean-0.1070.1780.000

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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 hetermotrime...

EntireName: Influenza A virus (A/NT/60/1968) polymerase dimer of hetermotrimer in complex with 3'5' cRNA promoter and Nb8205
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: 8

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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 hetermotrimer in complex with 3'5' cRNA promoter and Nb8205
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

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Component #2: protein, Influenza A virus (A/NT/60/1968) polymerase dimer of het...

ProteinName: Influenza A virus (A/NT/60/1968) polymerase dimer of hetermotrimer in complex with 3'5' cRNA promoter and Nb8205
Recombinant expression: No
SourceSpecies: Influenza A virus (A/nt/60/1968(H3N2))
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #3: protein, Influenza A virus (A/NT/60/1968) polymerase dimer of het...

ProteinName: Influenza A virus (A/NT/60/1968) polymerase dimer of hetermotrimer in complex with 3'5' cRNA promoter and Nb8205
Recombinant expression: No
SourceSpecies: Influenza A virus (A/nt/60/1968(H3N2))
Source (engineered)Expression System: synthetic construct (others)

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Component #4: protein, Nb8205

ProteinName: Nb8205 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 14.835375 kDa
SourceSpecies: Influenza A virus (A/nt/60/1968(H3N2))
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #5: protein, Polymerase acidic protein

ProteinName: Polymerase acidic protein / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 83.100797 kDa
SourceSpecies: Influenza A virus (A/nt/60/1968(H3N2))
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #6: protein, RNA-directed RNA polymerase catalytic subunit

ProteinName: RNA-directed RNA polymerase catalytic subunit / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 86.524086 kDa
SourceSpecies: Influenza A virus (A/nt/60/1968(H3N2))
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #7: protein, Polymerase basic protein 2

ProteinName: Polymerase basic protein 2 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 86.163391 kDa
SourceSpecies: Influenza A virus (A/nt/60/1968(H3N2))
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #8: nucleic-acid, RNA (5'-R(P*AP*GP*CP*AP*AP*AP*AP*GP*CP*AP*GP*G)-3')

nucleic acidName: RNA (5'-R(P*AP*GP*CP*AP*AP*AP*AP*GP*CP*AP*GP*G)-3') / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
PAGCAAAAGC AGGCC
MassTheoretical: 4.901097 kDa
SourceSpecies: Influenza A virus (A/nt/60/1968(H3N2))

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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: 1.25 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: 2456

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 27861
3D reconstructionSoftware: RELION / Resolution: 4.15 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Refinement space: REAL / Details: Rigid body fit only
Input PDB model: 6QX8, 6QX8, 6QX8, 6QX8, 6QPG, 6QPG
Chain ID: A, B, C, D, M, O

Overall bvalue: 95
Output model

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