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- EMDB-10019: RNA Polymerase I Open Complex conformation 2 focused refinement on CF -

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Basic information

Entry
Database: EMDB / ID: EMD-10019
TitleRNA Polymerase I Open Complex conformation 2 focused refinement on CF
Map dataRNA Polymerase I Open Complex conformation 2 (OC2) focused refinement on CF
Sample
  • Complex: RNA Polymerase I Open Complex conformation 2 focused refinement on CF
    • Complex: RNA Polymerase I
    • Complex: DNA
    • Complex: transcription initiation factors
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsMueller CW / Sadian Y
Funding support1 items
OrganizationGrant numberCountry
European Research CouncilERC-2013-AdG340964-POL1PIC
CitationJournal: Nat Commun / Year: 2019
Title: Molecular insight into RNA polymerase I promoter recognition and promoter melting.
Authors: Yashar Sadian / Florence Baudin / Lucas Tafur / Brice Murciano / Rene Wetzel / Felix Weis / Christoph W Müller /
Abstract: RNA polymerase I (Pol I) assembles with core factor (CF) and Rrn3 on the rDNA core promoter for transcription initiation. Here, we report cryo-EM structures of closed, intermediate and open Pol I ...RNA polymerase I (Pol I) assembles with core factor (CF) and Rrn3 on the rDNA core promoter for transcription initiation. Here, we report cryo-EM structures of closed, intermediate and open Pol I initiation complexes from 2.7 to 3.7 Å resolution to visualize Pol I promoter melting and to structurally and biochemically characterize the recognition mechanism of Pol I promoter DNA. In the closed complex, double-stranded DNA runs outside the DNA-binding cleft. Rotation of CF and upstream DNA with respect to Pol I and Rrn3 results in the spontaneous loading and opening of the promoter followed by cleft closure and positioning of the Pol I A49 tandem winged helix domain (tWH) onto DNA. Conformational rearrangement of A49 tWH leads to a clash with Rrn3 to initiate complex disassembly and promoter escape. Comprehensive insight into the Pol I transcription initiation cycle allows comparisons with promoter opening by Pol II and Pol III.
History
DepositionJun 3, 2019-
Header (metadata) releaseApr 22, 2020-
Map releaseApr 22, 2020-
UpdateApr 22, 2020-
Current statusApr 22, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10019.png

Downloads & links

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Map

FileDownload / File: emd_10019.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRNA Polymerase I Open Complex conformation 2 (OC2) focused refinement on CF
Voxel sizeX=Y=Z: 1.32 Å
Density
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.2007255 - 0.38596818
Average (Standard dev.)0.0001728115 (±0.005234903)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 380.16 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.321.321.32
M x/y/z288288288
origin x/y/z0.0000.0000.000
length x/y/z380.160380.160380.160
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS288288288
D min/max/mean-0.2010.3860.000

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Supplemental data

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Sample components

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Entire : RNA Polymerase I Open Complex conformation 2 focused refinement on CF

EntireName: RNA Polymerase I Open Complex conformation 2 focused refinement on CF
Components
  • Complex: RNA Polymerase I Open Complex conformation 2 focused refinement on CF
    • Complex: RNA Polymerase I
    • Complex: DNA
    • Complex: transcription initiation factors

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Supramolecule #1: RNA Polymerase I Open Complex conformation 2 focused refinement on CF

SupramoleculeName: RNA Polymerase I Open Complex conformation 2 focused refinement on CF
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#20

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Supramolecule #2: RNA Polymerase I

SupramoleculeName: RNA Polymerase I / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #3-#16
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: synthetic construct (others)

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Supramolecule #4: transcription initiation factors

SupramoleculeName: transcription initiation factors / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #17-#20
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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: 100 % / Chamber temperature: 4 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 1.57175 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 59963

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