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- EMDB-12723: Focused refinement of the upstream complex region of a RNA polyme... -

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Basic information

Entry
Database: EMDB / ID: EMD-12723
TitleFocused refinement of the upstream complex region of a RNA polymerase II transcription pre-initiation assembly
Map data
Sample
  • Complex: Yeast RNA polymerase II transcription pre-initiation complex
Biological speciesSaccharomyces cerevisiae S288C (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsSchilbach S / Aibara S / Dienemann C / Grabbe F / Cramer P
Funding support Germany, 5 items
OrganizationGrant numberCountry
H2020 Marie Curie Actions of the European Commission894862 Germany
German Research Foundation (DFG)EXC 2067/1 39072994 Germany
German Research Foundation (DFG)SFB860 Germany
German Research Foundation (DFG)SPP2191 Germany
European Research Council (ERC)882357 Germany
CitationJournal: Cell / Year: 2021
Title: Structure of RNA polymerase II pre-initiation complex at 2.9 Å defines initial DNA opening.
Authors: Sandra Schilbach / Shintaro Aibara / Christian Dienemann / Frauke Grabbe / Patrick Cramer /
Abstract: Transcription initiation requires assembly of the RNA polymerase II (Pol II) pre-initiation complex (PIC) and opening of promoter DNA. Here, we present the long-sought high-resolution structure of ...Transcription initiation requires assembly of the RNA polymerase II (Pol II) pre-initiation complex (PIC) and opening of promoter DNA. Here, we present the long-sought high-resolution structure of the yeast PIC and define the mechanism of initial DNA opening. We trap the PIC in an intermediate state that contains half a turn of open DNA located 30-35 base pairs downstream of the TATA box. The initially opened DNA region is flanked and stabilized by the polymerase "clamp head loop" and the TFIIF "charged region" that both contribute to promoter-initiated transcription. TFIIE facilitates initiation by buttressing the clamp head loop and by regulating the TFIIH translocase. The initial DNA bubble is then extended in the upstream direction, leading to the open promoter complex and enabling start-site scanning and RNA synthesis. This unique mechanism of DNA opening may permit more intricate regulation than in the Pol I and Pol III systems.
History
DepositionApr 6, 2021-
Header (metadata) releaseJun 16, 2021-
Map releaseJun 16, 2021-
UpdateAug 4, 2021-
Current statusAug 4, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 5
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12723.png

Downloads & links

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Map

FileDownload / File: emd_12723.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.05 Å/pix.
x 280 pix.
= 294. Å		1.05 Å/pix.
x 280 pix.
= 294. Å		1.05 Å/pix.
x 280 pix.
= 294. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.05 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 5.0 / Movie #1: 5
Minimum - Maximum-58.784714 - 121.28149
Average (Standard dev.)-4.6612263e-12 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 294.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z294.000294.000294.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ280280280
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS280280280
D min/max/mean-58.785121.281-0.000

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Supplemental data

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Mask #1

Fileemd_12723_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_12723_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_12723_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Yeast RNA polymerase II transcription pre-initiation complex

EntireName: Yeast RNA polymerase II transcription pre-initiation complex
Components
  • Complex: Yeast RNA polymerase II transcription pre-initiation complex

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Supramolecule #1: Yeast RNA polymerase II transcription pre-initiation complex

SupramoleculeName: Yeast RNA polymerase II transcription pre-initiation complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#30
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightTheoretical: 1.37 MDa

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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: 7.6
GridModel: Quantifoil R3.5/1 / Material: COPPER / Support film - Material: CARBON / Support film - topology: CONTINUOUS
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 29670 / Average exposure time: 9.0 sec. / Average electron dose: 43.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 130000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4300000
CTF correctionSoftware: (Name: Warp, RELION)
Startup modelType of model: EMDB MAP
EMDB ID:

3846

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 156629
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationSoftware - Name: RELION
FSC plot (resolution estimation)

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