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- EMDB-3443: Electron cryo-microscopy of the yeast RNA polymerase I - Rrn3 com... -

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Basic information

Entry
Database: EMDB / ID: EMD-3443
TitleElectron cryo-microscopy of the yeast RNA polymerase I - Rrn3 complex at 7.5A resolution
Map datareconstruction of the RNA pol I - Rrn3 complex
Sample
  • Sample: complex formed between YEAST RNA polymerase I and the initiation factor Rrn3
  • Protein or peptide: DNA dependent RNA polymerase I
  • Protein or peptide: Rrn3
KeywordsRNA polymerase I initiation factor Rrn3 transcription initiation
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 7.5 Å
AuthorsPilsl M / Crucifix C / Papai G / Krupp F / Steinbauer R / Griesenbeck J / Milkereit P / Tschochner H / Schultz P
CitationJournal: Nat Commun / Year: 2016
Title: Structure of the initiation-competent RNA polymerase I and its implication for transcription.
Authors: Michael Pilsl / Corinne Crucifix / Gabor Papai / Ferdinand Krupp / Robert Steinbauer / Joachim Griesenbeck / Philipp Milkereit / Herbert Tschochner / Patrick Schultz /
Abstract: Eukaryotic RNA polymerase I (Pol I) is specialized in rRNA gene transcription synthesizing up to 60% of cellular RNA. High level rRNA production relies on efficient binding of initiation factors to ...Eukaryotic RNA polymerase I (Pol I) is specialized in rRNA gene transcription synthesizing up to 60% of cellular RNA. High level rRNA production relies on efficient binding of initiation factors to the rRNA gene promoter and recruitment of Pol I complexes containing initiation factor Rrn3. Here, we determine the cryo-EM structure of the Pol I-Rrn3 complex at 7.5 Å resolution, and compare it with Rrn3-free monomeric and dimeric Pol I. We observe that Rrn3 contacts the Pol I A43/A14 stalk and subunits A190 and AC40, that association re-organizes the Rrn3 interaction interface, thereby preventing Pol I dimerization; and Rrn3-bound and monomeric Pol I differ from the dimeric enzyme in cleft opening, and localization of the A12.2 C-terminus in the active centre. Our findings thus support a dual role for Rrn3 in transcription initiation to stabilize a monomeric initiation competent Pol I and to drive pre-initiation complex formation.
History
DepositionJul 21, 2016-
Header (metadata) releaseSep 28, 2016-
Map releaseSep 28, 2016-
UpdateOct 12, 2016-
Current statusOct 12, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.121
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.121
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

image.png

Downloads & links

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Map

FileDownload / File: emd_3443.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationreconstruction of the RNA pol I - Rrn3 complex
Voxel sizeX=Y=Z: 2.16 Å
Density
Contour LevelBy AUTHOR: 0.121 / Movie #1: 0.121
Minimum - Maximum-0.17636499 - 0.38812745
Average (Standard dev.)0.00050278 (±0.02722943)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.162.162.16
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z276.480276.480276.480
α/β/γ90.00090.00090.000
start NX/NY/NZ-147-147-146
NX/NY/NZ294294294
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.1760.3880.001

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Supplemental data

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Sample components

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Entire : complex formed between YEAST RNA polymerase I and the initiation ...

EntireName: complex formed between YEAST RNA polymerase I and the initiation factor Rrn3
Components
  • Sample: complex formed between YEAST RNA polymerase I and the initiation factor Rrn3
  • Protein or peptide: DNA dependent RNA polymerase I
  • Protein or peptide: Rrn3

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Supramolecule #1000: complex formed between YEAST RNA polymerase I and the initiation ...

SupramoleculeName: complex formed between YEAST RNA polymerase I and the initiation factor Rrn3
type: sample / ID: 1000 / Oligomeric state: 1 / Number unique components: 2
Molecular weightTheoretical: 663 MDa

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Macromolecule #1: DNA dependent RNA polymerase I

MacromoleculeName: DNA dependent RNA polymerase I / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: bakers yeast / Location in cell: nucleolar
Molecular weightTheoretical: 590 KDa

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Macromolecule #2: Rrn3

MacromoleculeName: Rrn3 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: bakers yeast / Location in cell: nucleolar
Molecular weightTheoretical: 72.346 KDa

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.01 mg/mL
BufferpH: 7.8 / Details: 20 mM Hepes, 100mM ammonium acetate and 2mM MgCl2
StainingType: NEGATIVE / Details: unstained
GridDetails: sample was adsorbed on a floated carbon foil which was deposited onto a quantifoil grid
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Instrument: FEI VITROBOT MARK IV / Method: blotting time (4 sec.) blotting force 5

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 129630 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.01 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
TemperatureMin: 80 K / Max: 100 K / Average: 90 K
Alignment procedureLegacy - Astigmatism: Cs corrector
DateJun 10, 2015
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Average electron dose: 22 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 7.5 Å / Resolution method: OTHER / Software - Name: relion / Details: final map was calculated from 2 averaged datasets / Number images used: 32438

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Atomic model buiding 1

Initial modelPDB ID:

4c2m

SoftwareName: gEMfitter, Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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