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- EMDB-10663: Focused refinement CF in eiPIC -

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Basic information

Entry
Database: EMDB / ID: EMD-10663
TitleFocused refinement CF in eiPIC
Map data
Sample
  • Complex: early intermediate RNA-Polymerase I Pre-initiation Complex - eiPIC
    • Complex: RNA polymerase
    • Complex: transcription initiation factor
    • Complex: DNA
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) / Synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.54 Å
AuthorsPilsl M / Engel C
Funding support Germany, 2 items
OrganizationGrant numberCountry
German Research Foundation (DFG)EN 1204/1-1 Germany
German Research Foundation (DFG)CRC 960 Germany
CitationJournal: Nat Commun / Year: 2020
Title: Structural basis of RNA polymerase I pre-initiation complex formation and promoter melting.
Authors: Michael Pilsl / Christoph Engel /
Abstract: Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a prerequisite for the biosynthesis of ribosomes in eukaryotes. Compared to Pols II and III, the mechanisms underlying ...Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a prerequisite for the biosynthesis of ribosomes in eukaryotes. Compared to Pols II and III, the mechanisms underlying promoter recognition, initiation complex formation and DNA melting by Pol I substantially diverge. Here, we report the high-resolution cryo-EM reconstruction of a Pol I early initiation intermediate assembled on a double-stranded promoter scaffold that prevents the establishment of downstream DNA contacts. Our analyses demonstrate how efficient promoter-backbone interaction is achieved by combined re-arrangements of flexible regions in the 'core factor' subunits Rrn7 and Rrn11. Furthermore, structure-function analysis illustrates how destabilization of the melted DNA region correlates with contraction of the polymerase cleft upon transcription activation, thereby combining promoter recruitment with DNA-melting. This suggests that molecular mechanisms and structural features of Pol I initiation have co-evolved to support the efficient melting, initial transcription and promoter clearance required for high-level rRNA synthesis.
History
DepositionFeb 4, 2020-
Header (metadata) releaseMar 18, 2020-
Map releaseMar 18, 2020-
UpdateMar 18, 2020-
Current statusMar 18, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.022
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.022
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10663.png

Downloads & links

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Map

FileDownload / File: emd_10663.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.09 Å/pix.
x 420 pix.
= 457.8 Å		1.09 Å/pix.
x 420 pix.
= 457.8 Å		1.09 Å/pix.
x 420 pix.
= 457.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.09 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.022 / Movie #1: 0.022
Minimum - Maximum-0.030239038 - 0.05790723
Average (Standard dev.)-0.00003387238 (±0.0013648649)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 457.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.091.091.09
M x/y/z420420420
origin x/y/z0.0000.0000.000
length x/y/z457.800457.800457.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS420420420
D min/max/mean-0.0300.058-0.000

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Supplemental data

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Sample components

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Entire : early intermediate RNA-Polymerase I Pre-initiation Complex - eiPIC

EntireName: early intermediate RNA-Polymerase I Pre-initiation Complex - eiPIC
Components
  • Complex: early intermediate RNA-Polymerase I Pre-initiation Complex - eiPIC
    • Complex: RNA polymerase
    • Complex: transcription initiation factor
    • Complex: DNA

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Supramolecule #1: early intermediate RNA-Polymerase I Pre-initiation Complex - eiPIC

SupramoleculeName: early intermediate RNA-Polymerase I Pre-initiation Complex - eiPIC
type: complex / ID: 1 / Parent: 0

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Supramolecule #2: RNA polymerase

SupramoleculeName: RNA polymerase / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Supramolecule #3: transcription initiation factor

SupramoleculeName: transcription initiation factor / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #4: DNA

SupramoleculeName: DNA / type: complex / ID: 4 / Parent: 1
Source (natural)Organism: Synthetic construct (others)
Recombinant expressionOrganism: Synthetic construct (others)

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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

Concentration0.1 mg/mL
BufferpH: 7.8 / Component - Formula: KCl
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 4088 / Average electron dose: 1.4 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.01 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: RELION (ver. 3.0) / Software - details: 3
Startup modelType of model: NONE / Details: Initial model generated from 2D classes
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.54 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.08) / Number images used: 122099
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0) / Software - details: 3
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0) / Software - details: 3

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