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- EMDB-9581: Structure of influenza D virus polymerase bound to vRNA promoter ... -

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Basic information

Entry
Database: EMDB / ID: EMD-9581
TitleStructure of influenza D virus polymerase bound to vRNA promoter in Mode B conformation (Class B1)
Map data
SampleInfluenza D virus polymerase bound to vRNA promoter
  • (nucleic-acidNucleic acid) x 2
  • Polymerase 3
  • RNA-directed RNA polymerase catalytic subunit
  • Polymerase PB2
Function / homology
Function and homology information


viral RNA genome replication / RNA-directed RNA polymerase / RNA-directed 5'-3' RNA polymerase activity / RNA binding
Influenza RNA-dependent RNA polymerase subunit PA / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB2 / RNA-directed RNA polymerase, negative-strand RNA virus / PA/PA-X superfamily
RNA-directed RNA polymerase catalytic subunit / Polymerase PB2 / Polymerase 3
Biological speciesInfluenza D virus / synthetic construct (others) / Influenza D virus (D/swine/Oklahoma/1334/2011)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsPeng Q / Peng R / Qi J / Gao GF / Shi Y
Funding support China, 1 items
OrganizationGrant numberCountry
Chinese Academy of SciencesXDB08020100 China
CitationJournal: Nat Microbiol / Year: 2019
Title: Structural insight into RNA synthesis by influenza D polymerase.
Authors: Qi Peng / Yuqian Liu / Ruchao Peng / Min Wang / Wei Yang / Hao Song / Yuhai Chen / Sheng Liu / Min Han / Xinzheng Zhang / Peiyi Wang / Jinghua Yan / Buchang Zhang / Jianxun Qi / Tao Deng / ...Authors: Qi Peng / Yuqian Liu / Ruchao Peng / Min Wang / Wei Yang / Hao Song / Yuhai Chen / Sheng Liu / Min Han / Xinzheng Zhang / Peiyi Wang / Jinghua Yan / Buchang Zhang / Jianxun Qi / Tao Deng / George F Gao / Yi Shi /
Abstract: The influenza virus polymerase uses capped RNA primers to initiate transcription, and a combination of terminal and internal de novo initiations for the two-step replication process by binding the ...The influenza virus polymerase uses capped RNA primers to initiate transcription, and a combination of terminal and internal de novo initiations for the two-step replication process by binding the conserved viral genomic RNA (vRNA) or complementary RNA (cRNA) promoter. Here, we determined the apo and promoter-bound influenza D polymerase structures using cryo-electron microscopy and found the polymerase has an evolutionarily conserved stable core structure with inherently flexible peripheral domains. Strikingly, two conformations (mode A and B) of the vRNA promoter were observed where the 3'-vRNA end can bind at two different sites, whereas the cRNA promoter only binds in the mode B conformation. Functional studies confirmed the critical role of the mode B conformation for vRNA synthesis via the intermediate cRNA but not for cRNA production, which is mainly regulated by the mode A conformation. Both conformations participate in the regulation of the transcription process. This work advances our understanding of the regulatory mechanisms for the synthesis of different RNA species by influenza virus polymerase and opens new opportunities for antiviral drug design.
Validation ReportPDB-ID: 6kur

SummaryFull reportAbout validation report
History
DepositionJul 21, 2018-
Header (metadata) releaseJun 12, 2019-
Map releaseJun 12, 2019-
UpdateFeb 12, 2020-
Current statusFeb 12, 2020Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6kur
  • Surface level: 0.05
  • 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_9581.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.36 Å/pix.
x 180 pix.
= 244.8 Å
1.36 Å/pix.
x 180 pix.
= 244.8 Å
1.36 Å/pix.
x 180 pix.
= 244.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.36 Å
Density
Contour LevelBy AUTHOR: 0.043 / Movie #1: 0.05
Minimum - Maximum-0.24586575 - 0.40841785
Average (Standard dev.)0.0005774942 (±0.011223717)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 244.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.361.361.36
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z244.800244.800244.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS180180180
D min/max/mean-0.2460.4080.001

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Supplemental data

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Sample components

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Entire Influenza D virus polymerase bound to vRNA promoter

EntireName: Influenza D virus polymerase bound to vRNA promoter / Number of components: 6

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Component #1: protein, Influenza D virus polymerase bound to vRNA promoter

ProteinName: Influenza D virus polymerase bound to vRNA promoter / Recombinant expression: No
MassExperimental: 270 kDa
SourceSpecies: Influenza D virus
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm) / Cell of expression system: SF9

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Component #2: nucleic-acid, RNA (5'-R(P*CP*UP*CP*CP*UP*GP*CP*UP*UP*AP*UP*GP*CP*...

nucleic acidName: RNA (5'-R(P*CP*UP*CP*CP*UP*GP*CP*UP*UP*AP*UP*GP*CP*U)-3')
Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
CUCCUGCUUA UGCU
MassTheoretical: 4.337563 kDa
SourceSpecies: synthetic construct (others)

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Component #3: nucleic-acid, RNA (5'-R(P*AP*GP*CP*AP*GP*UP*AP*GP*CP*AP*AP*GP*GP*...

nucleic acidName: RNA (5'-R(P*AP*GP*CP*AP*GP*UP*AP*GP*CP*AP*AP*GP*GP*AP*G)-3')
Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
AGCAGUAGCA AGGAG
MassTheoretical: 4.918042 kDa
SourceSpecies: synthetic construct (others)

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Component #4: protein, Polymerase 3

ProteinName: Polymerase 3 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 83.036086 kDa
SourceSpecies: Influenza D virus (D/swine/Oklahoma/1334/2011) / Strain: D/swine/Oklahoma/1334/2011
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #5: protein, RNA-directed RNA polymerase catalytic subunit

ProteinName: RNA-directed RNA polymerase catalytic subunit / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 86.138844 kDa
SourceSpecies: Influenza D virus (D/swine/Oklahoma/1334/2011) / Strain: D/swine/Oklahoma/1334/2011
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #6: protein, Polymerase PB2

ProteinName: Polymerase PB2 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 29.338217 kDa
SourceSpecies: Influenza D virus (D/swine/Oklahoma/1334/2011) / Strain: D/swine/Oklahoma/1334/2011
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.: 0.8 mg/mL / pH: 7.5
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %

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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: 60 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 QUANTUM (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 298468
3D reconstructionSoftware: RELION / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Refinement space: REAL
Output model

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