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- EMDB-6404: In situ structures of the segmented genome and RNA polymerase com... -

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Basic information

Entry
Database: EMDB / ID: EMD-6404
TitleIn situ structures of the segmented genome and RNA polymerase complex inside a dsRNA virus
Map dataCPV in the transcribing state
Sample
  • Sample: CPV VP4 + polymerase complex (TEC)
  • Protein or peptide: VP4
  • Protein or peptide: RNA polymerase
Keywordscryo-EM / dsRNA genome organization / viral polymerase
Function / homology
Function and homology information


viral genome replication / RNA-directed RNA polymerase / RNA-directed RNA polymerase activity / RNA binding
Similarity search - Function
: / : / CPV, RNA-directed RNA polymerase, N-terminal / CPV, RNA-directed RNA polymerase, central polymerase domain / CPV, RNA-directed RNA polymerase, C-terminal / : / CPV Capsid shell protein VP1, small protrusion domain / : / Inner layer core protein VP1-like, C-terminal / RNA-directed RNA polymerase, reovirus / RdRp of Reoviridae dsRNA viruses catalytic domain profile.
Similarity search - Domain/homology
RNA-directed RNA polymerase / VP1 / Viral structural protein 4
Similarity search - Component
Biological speciesBombyx mori cypovirus 1
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsZhang X / Ding K / Yu XK / Chang W / Sun JC / Zhou ZH
CitationJournal: Nature / Year: 2015
Title: In situ structures of the segmented genome and RNA polymerase complex inside a dsRNA virus.
Authors: Xing Zhang / Ke Ding / Xuekui Yu / Winston Chang / Jingchen Sun / Z Hong Zhou /
Abstract: Viruses in the Reoviridae, like the triple-shelled human rotavirus and the single-shelled insect cytoplasmic polyhedrosis virus (CPV), all package a genome of segmented double-stranded RNAs (dsRNAs) ...Viruses in the Reoviridae, like the triple-shelled human rotavirus and the single-shelled insect cytoplasmic polyhedrosis virus (CPV), all package a genome of segmented double-stranded RNAs (dsRNAs) inside the viral capsid and carry out endogenous messenger RNA synthesis through a transcriptional enzyme complex (TEC). By direct electron-counting cryoelectron microscopy and asymmetric reconstruction, we have determined the organization of the dsRNA genome inside quiescent CPV (q-CPV) and the in situ atomic structures of TEC within CPV in both quiescent and transcribing (t-CPV) states. We show that the ten segmented dsRNAs in CPV are organized with ten TECs in a specific, non-symmetric manner, with each dsRNA segment attached directly to a TEC. The TEC consists of two extensively interacting subunits: an RNA-dependent RNA polymerase (RdRP) and an NTPase VP4. We find that the bracelet domain of RdRP undergoes marked conformational change when q-CPV is converted to t-CPV, leading to formation of the RNA template entry channel and access to the polymerase active site. An amino-terminal helix from each of two subunits of the capsid shell protein (CSP) interacts with VP4 and RdRP. These findings establish the link between sensing of environmental cues by the external proteins and activation of endogenous RNA transcription by the TEC inside the virus.
History
DepositionAug 1, 2015-
Header (metadata) releaseAug 19, 2015-
Map releaseOct 28, 2015-
UpdateDec 2, 2015-
Current statusDec 2, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jb7
  • Surface level: 1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6404.jpg

Downloads & links

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Map

FileDownload / File: emd_6404.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCPV in the transcribing state
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
1.36 Å/pix.
x 256 pix.
= 348.16 Å		1.36 Å/pix.
x 256 pix.
= 348.16 Å		1.36 Å/pix.
x 256 pix.
= 348.16 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

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.2 / Movie #1: 1
Minimum - Maximum-2.31748009 - 4.60761023
Average (Standard dev.)0.00443179 (±0.09505297)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-35-13026
Dimensions256256256
Spacing256256256
CellA=B=C: 348.16 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.361.361.36
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z348.160348.160348.160
α/β/γ90.00090.00090.000
start NX/NY/NZ-35-13026
NX/NY/NZ256256256
MAP C/R/S213
start NC/NR/NS-130-3526
NC/NR/NS256256256
D min/max/mean-2.3174.6080.004

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Supplemental data

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Sample components

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Entire : CPV VP4 + polymerase complex (TEC)

EntireName: CPV VP4 + polymerase complex (TEC)
Components
  • Sample: CPV VP4 + polymerase complex (TEC)
  • Protein or peptide: VP4
  • Protein or peptide: RNA polymerase

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Supramolecule #1000: CPV VP4 + polymerase complex (TEC)

SupramoleculeName: CPV VP4 + polymerase complex (TEC) / type: sample / ID: 1000 / Number unique components: 2

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Macromolecule #1: VP4

MacromoleculeName: VP4 / type: protein_or_peptide / ID: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bombyx mori cypovirus 1 / synonym: Cytoplasmic polyhedrosis virus

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Macromolecule #2: RNA polymerase

MacromoleculeName: RNA polymerase / type: protein_or_peptide / ID: 2 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bombyx mori cypovirus 1 / synonym: Cytoplasmic polyhedrosis 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

BufferpH: 8 / Details: 70 mM Tris-Cl, 10 mM MgCl2, 100 mM NaCl, 2 mM GTP
GridDetails: 200 mesh Quantifoil holey carbon film
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK II

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureAverage: 80 K
Alignment procedureLegacy - Electron beam tilt params: 0
Specialist opticsEnergy filter - Name: Gatan Quantum Energy Filter
DateMar 7, 2015
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Digitization - Sampling interval: 5 µm / Number real images: 4907 / Average electron dose: 40 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 36765 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.4 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: OTHER / Software - Name: Frealign / Number images used: 81887
Final two d classificationNumber classes: 1

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