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- EMDB-6203: Electron cryo-microscopy of an RNA polymerase -

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Basic information

Entry
Database: EMDB / ID: EMD-6203
TitleElectron cryo-microscopy of an RNA polymerase
Map dataReconstruction of ter
Sample
  • Sample: tetrameric Influenza Virus RNA Polymerase
  • Protein or peptide: Influenza Virus RNA Polymerase Complex
KeywordsInfluenza RdRP / single particle reconstitution / replication
Function / homology
Function and homology information


cap snatching / viral transcription / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / endonuclease activity / host cell cytoplasm / Hydrolases; Acting on ester bonds / RNA-directed RNA polymerase / viral translational frameshifting / viral RNA genome replication / RNA-dependent RNA polymerase activity ...cap snatching / viral transcription / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / endonuclease activity / host cell cytoplasm / Hydrolases; Acting on ester bonds / RNA-directed RNA polymerase / viral translational frameshifting / viral RNA genome replication / RNA-dependent RNA polymerase activity / nucleotide binding / DNA-templated transcription / host cell nucleus / RNA binding / identical protein binding / metal ion binding
Similarity search - Function
Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB1 / 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 acidic protein / RNA-directed RNA polymerase catalytic subunit
Similarity search - Component
Biological speciesInfluenza A virus
Methodsingle particle reconstruction / cryo EM / Resolution: 6.8 Å
AuthorsChang S / Sun DP / Liang H / Wang J / Li J / Guo L / Wang X / Guan C / Boruah BM / Yuan L ...Chang S / Sun DP / Liang H / Wang J / Li J / Guo L / Wang X / Guan C / Boruah BM / Yuan L / Feng F / Yang M / Wojdyla J / Wang M / Wang HW / Liu Y
CitationJournal: Mol Cell / Year: 2015
Title: Cryo-EM structure of influenza virus RNA polymerase complex at 4.3 Å resolution.
Authors: Shenghai Chang / Dapeng Sun / Huanhuan Liang / Jia Wang / Jun Li / Lu Guo / Xiangli Wang / Chengcheng Guan / Bhargavi M Boruah / Lingmin Yuan / Feng Feng / Mingrui Yang / Lulan Wang / Yao ...Authors: Shenghai Chang / Dapeng Sun / Huanhuan Liang / Jia Wang / Jun Li / Lu Guo / Xiangli Wang / Chengcheng Guan / Bhargavi M Boruah / Lingmin Yuan / Feng Feng / Mingrui Yang / Lulan Wang / Yao Wang / Justyna Wojdyla / Lanjuan Li / Jiawei Wang / Meitian Wang / Genhong Cheng / Hong-Wei Wang / Yingfang Liu /
Abstract: Replication and transcription of influenza virus genome mainly depend on its RNA-dependent RNA polymerase (RdRP), composed of the PA, PB1, and PB2 subunits. Although extensively studied, the ...Replication and transcription of influenza virus genome mainly depend on its RNA-dependent RNA polymerase (RdRP), composed of the PA, PB1, and PB2 subunits. Although extensively studied, the underlying mechanism of the RdRP complex is still unclear. Here we report the biochemical characterization of influenza RdRP subcomplex comprising PA, PB1, and N terminus of PB2, which exist as dimer in solution and can assemble into a tetramer state, regulated by vRNA promoter. Using single-particle cryo-electron microscopy, we have reconstructed the RdRP tetramer complex at 4.3 Å, highlighting the assembly and interfaces between monomers within the tetrameric structure. The individual RdRP subcomplex contains all the characterized motifs and appears as a cage-like structure. High-throughput mutagenesis profiling revealed that residues involved in the oligomer state formation are critical for viral life cycle. Our results lay a solid base for understanding the mechanism of replication of influenza and other negative-stranded RNA viruses.
History
DepositionNov 28, 2014-
Header (metadata) releaseDec 31, 2014-
Map releaseFeb 4, 2015-
UpdateJun 3, 2015-
Current statusJun 3, 2015Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.018
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.018
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6203.gif

Downloads & links

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Map

FileDownload / File: emd_6203.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of ter
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.32 Å/pix.
x 256 pix.
= 337.92 Å		1.32 Å/pix.
x 256 pix.
= 337.92 Å		1.32 Å/pix.
x 256 pix.
= 337.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.32 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.018 / Movie #1: 0.018
Minimum - Maximum-0.02080948 - 0.06722944
Average (Standard dev.)0.00058243 (±0.00367799)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 337.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.321.321.32
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z337.920337.920337.920
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0210.0670.001

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Supplemental data

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Sample components

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Entire : tetrameric Influenza Virus RNA Polymerase

EntireName: tetrameric Influenza Virus RNA Polymerase
Components
  • Sample: tetrameric Influenza Virus RNA Polymerase
  • Protein or peptide: Influenza Virus RNA Polymerase Complex

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Supramolecule #1000: tetrameric Influenza Virus RNA Polymerase

SupramoleculeName: tetrameric Influenza Virus RNA Polymerase / type: sample / ID: 1000 / Number unique components: 1

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Macromolecule #1: Influenza Virus RNA Polymerase Complex

MacromoleculeName: Influenza Virus RNA Polymerase Complex / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Oligomeric state: tetramer / Recombinant expression: Yes / Database: NCBI
Source (natural)Organism: Influenza A virus

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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

Concentration0.2 mg/mL
BufferpH: 7.8 / Details: 25 mM HEPES 200 mM NaCl and 1 mM EDTA
GridDetails: Quantifoil Cu R2/1
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV
Method: The grids were blotted for 6.5 s with 100% humidity and flash frozen in liquid ethane cooled at liquid nitrogen temperature using an FEI Vitrobot.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DateOct 2, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Average electron dose: 50 e/Å2
Details: Each image was fractionated into 32 sub-frames, with 0.25 s exposure time per frame. All dose-fractionated cryo-EM images were recorded using a semi-automated low-dose acquisition program UCSF-Image4
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.8 Å / Resolution method: OTHER / Software - Name: EMAN2, RELION / Number images used: 67066

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Atomic model buiding 1

Initial modelPDB ID:

3cm8

SoftwareName: chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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