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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 data
Samplecomplex formed between YEAST RNA polymerase I and the initiation factor Rrn3:
DNA dependent RNA polymerase I / Rrn3
KeywordsRNA polymerase I initiation factor Rrn3 transcription initiation
Biological speciesSaccharomyces cerevisiae (baker'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
Current status-Processing site: PDBe / Status: Released
DepositionJul 21, 2016-
Header (metadata) releaseSep 28, 2016-
Map releaseSep 28, 2016-
UpdateOct 12, 2016-

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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
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

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)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.16 Å/pix.
x 128 pix.
= 276.48 Å
2.16 Å/pix.
x 128 pix.
= 276.48 Å
2.16 Å/pix.
x 128 pix.
= 276.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

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
Number of components: 2 / Oligomeric State: 1
MassTheoretical: 663 MDa

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Component #1: protein, DNA dependent RNA polymerase I

ProteinName: DNA dependent RNA polymerase I / Oligomeric Details: monomer / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 590 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (natural)Location in cell: nucleolar

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Component #2: protein, Rrn3

ProteinName: Rrn3 / Oligomeric Details: monomer / Recombinant expression: No / Number of Copies: 1
MassTheoretical: 72.346 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (natural)Location in cell: nucleolar

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: negative staining, cryo EM
Sample solutionSpecimen conc.: 0.01 mg/mL
Buffer solution: 20 mM Hepes, 100mM ammonium acetate and 2mM MgCl2
pH: 7.8
Support filmsample was adsorbed on a floated carbon foil which was deposited onto a quantifoil grid
Stainingunstained
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Method: blotting time (4 sec.) blotting force 5

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Jun 10, 2015
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 22 e/Å2 / Illumination mode: SPOT SCAN
LensMagnification: 59000 X (nominal), 129630 X (calibrated) / Astigmatism: Cs corrector / Cs: 0.01 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500 - 3500 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: 90 (80 - 100 K)
CameraDetector: FEI FALCON II (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 32438
3D reconstructionAlgorithm: relion / Software: relion / CTF correction: each particle / Details: final map was calculated from 2 averaged datasets / Resolution: 7.5 Å / Resolution method: FSC 0.143, gold-standard

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Atomic model buiding

Modeling #1Software: gEMfitter, Chimera / Refinement protocol: rigid body / Refinement space: REAL
Input PDB model: 4C2M

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