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- EMDB-4891: Structural basis of Poxvirus transcription: transcribing and capp... -

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Basic information

Entry
Database: EMDB / ID: EMD-4891
TitleStructural basis of Poxvirus transcription: transcribing and capping complexes (nucleic acid free particle population)
Map dataPost-processed map.
Sample
  • Complex: Vaccinia Virus DNA-dependent RNA polymerase co-transcriptional capping complex
Biological speciesVaccinia virus
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsHillen HS / Bartuli J / Grimm C / Dienemann C / Bedenk K / Szalay A / Fischer U / Cramer P
Funding support Germany, 7 items
OrganizationGrant numberCountry
German Research FoundationSFB860 Germany
German Federal Ministry for Education and ResearchEXC 2067/1- 390729940 Germany
German Research FoundationSPP1935 Germany
Volkswagen Foundation Germany
European Research CouncilTRANSREGULON Germany
German Research FoundationFi 573 18-1 Germany
German Research FoundationFi 573 7-2 Germany
CitationJournal: Cell / Year: 2019
Title: Structural Basis of Poxvirus Transcription: Transcribing and Capping Vaccinia Complexes.
Authors: Hauke S Hillen / Julia Bartuli / Clemens Grimm / Christian Dienemann / Kristina Bedenk / Aladar A Szalay / Utz Fischer / Patrick Cramer /
Abstract: Poxviruses use virus-encoded multisubunit RNA polymerases (vRNAPs) and RNA-processing factors to generate mG-capped mRNAs in the host cytoplasm. In the accompanying paper, we report structures of ...Poxviruses use virus-encoded multisubunit RNA polymerases (vRNAPs) and RNA-processing factors to generate mG-capped mRNAs in the host cytoplasm. In the accompanying paper, we report structures of core and complete vRNAP complexes of the prototypic Vaccinia poxvirus (Grimm et al., 2019; in this issue of Cell). Here, we present the cryo-electron microscopy (cryo-EM) structures of Vaccinia vRNAP in the form of a transcribing elongation complex and in the form of a co-transcriptional capping complex that contains the viral capping enzyme (CE). The trifunctional CE forms two mobile modules that bind the polymerase surface around the RNA exit tunnel. RNA extends from the vRNAP active site through this tunnel and into the active site of the CE triphosphatase. Structural comparisons suggest that growing RNA triggers large-scale rearrangements on the surface of the transcription machinery during the transition from transcription initiation to RNA capping and elongation. Our structures unravel the basis for synthesis and co-transcriptional modification of poxvirus RNA.
History
DepositionApr 23, 2019-
Header (metadata) releaseDec 18, 2019-
Map releaseDec 18, 2019-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.044
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.044
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4891.png

Downloads & links

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Map

FileDownload / File: emd_4891.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPost-processed map.
Voxel sizeX=Y=Z: 1.05 Å
Density
Contour LevelBy AUTHOR: 0.044 / Movie #1: 0.044
Minimum - Maximum-0.07866412 - 0.14399342
Average (Standard dev.)0.00044788225 (±0.0044892225)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions340340340
Spacing340340340
CellA=B=C: 356.99997 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z340340340
origin x/y/z0.0000.0000.000
length x/y/z357.000357.000357.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ320320320
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS340340340
D min/max/mean-0.0790.1440.000

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Supplemental data

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Mask #1

Fileemd_4891_msk_1.map
Projections & Slices
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Additional map: 3D Refinement map.

Fileemd_4891_additional.map
Annotation3D Refinement map.
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Additional map: 3D Refinement map.

Fileemd_4891_additional_1.map
Annotation3D Refinement map.
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AxesZYX

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Half map: #2

Fileemd_4891_half_map_1.map
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Half map: #1

Fileemd_4891_half_map_2.map
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Sample components

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Entire : Vaccinia Virus DNA-dependent RNA polymerase co-transcriptional ca...

EntireName: Vaccinia Virus DNA-dependent RNA polymerase co-transcriptional capping complex
Components
  • Complex: Vaccinia Virus DNA-dependent RNA polymerase co-transcriptional capping complex

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Supramolecule #1: Vaccinia Virus DNA-dependent RNA polymerase co-transcriptional ca...

SupramoleculeName: Vaccinia Virus DNA-dependent RNA polymerase co-transcriptional capping complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#13
Source (natural)Organism: Vaccinia virus / Strain: 1h68
Recombinant expressionOrganism: Vaccinia virus / Recombinant strain: 1h439
Molecular weightTheoretical: 580 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

BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK II

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 4958 / Average electron dose: 1.02 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 632458
CTF correctionSoftware: (Name: RELION (ver. 3.0), Warp)
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio model generated by cryoSPARC.
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 12433
FSC plot (resolution estimation)

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