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- EMDB-1804: Conformational flexibility of RNA polymerase III during transcrip... -

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Basic information

Entry
Database: EMDB / ID: EMD-1804
TitleConformational flexibility of RNA polymerase III during transcriptional elongation
Map dataRNA Polymerase III at 10A
Sample
  • Sample: RNA Polymerase III
  • Protein or peptide: RNA polymerase III
KeywordsTranscription / RNA polymerase III / elongation complex / transcription initiation / transcription elongation / transcription termination
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 10.0 Å
AuthorsFernandez-Tornero C / Bottcher B / Rashid UJ / Steuerwald U / Florchinger B / Devos DP / Lindner D / Muller CW
CitationJournal: EMBO J / Year: 2010
Title: Conformational flexibility of RNA polymerase III during transcriptional elongation.
Authors: Carlos Fernández-Tornero / Bettina Böttcher / Umar Jan Rashid / Ulrich Steuerwald / Beate Flörchinger / Damien P Devos / Doris Lindner / Christoph W Müller /
Abstract: RNA polymerase (Pol) III is responsible for the transcription of genes encoding small RNAs, including tRNA, 5S rRNA and U6 RNA. Here, we report the electron cryomicroscopy structures of yeast Pol III ...RNA polymerase (Pol) III is responsible for the transcription of genes encoding small RNAs, including tRNA, 5S rRNA and U6 RNA. Here, we report the electron cryomicroscopy structures of yeast Pol III at 9.9 Å resolution and its elongation complex at 16.5 Å resolution. Particle sub-classification reveals prominent EM densities for the two Pol III-specific subcomplexes, C31/C82/C34 and C37/C53, that can be interpreted using homology models. While the winged-helix-containing C31/C82/C34 subcomplex initiates transcription from one side of the DNA-binding cleft, the C37/C53 subcomplex accesses the transcription bubble from the opposite side of this cleft. The transcribing Pol III enzyme structure not only shows the complete incoming DNA duplex, but also reveals the exit path of newly synthesized RNA. During transcriptional elongation, the Pol III-specific subcomplexes tightly enclose the incoming DNA duplex, which likely increases processivity and provides structural insights into the conformational switch between Pol III-mediated initiation and elongation.
History
DepositionOct 4, 2010-
Header (metadata) releaseOct 8, 2010-
Map releaseNov 1, 2010-
UpdateSep 9, 2011-
Current statusSep 9, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.4
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.4
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1804.png

Downloads & links

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Map

FileDownload / File: emd_1804.map.gz / Format: CCP4 / Size: 21.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRNA Polymerase III at 10A
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.4 Å/pix.
x 180 pix.
= 252. Å		1.4 Å/pix.
x 180 pix.
= 252. Å		1.4 Å/pix.
x 180 pix.
= 252. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.4 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.4 / Movie #1: 0.4
Minimum - Maximum-2.81566 - 3.5073
Average (Standard dev.)-0.00622229 (±0.324437)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 252 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.41.41.4
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z252.000252.000252.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-150-150-149
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS180180180
D min/max/mean-2.8163.507-0.006

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Supplemental data

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Sample components

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Entire : RNA Polymerase III

EntireName: RNA Polymerase III
Components
  • Sample: RNA Polymerase III
  • Protein or peptide: RNA polymerase III

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Supramolecule #1000: RNA Polymerase III

SupramoleculeName: RNA Polymerase III / type: sample / ID: 1000 / Details: Sample was monodisperse
Oligomeric state: RNA PolIII complex of 17 different polypeptides
Number unique components: 1
Molecular weightExperimental: 700 KDa / Theoretical: 700 KDa / Method: Native mass spectrometry

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Macromolecule #1: RNA polymerase III

MacromoleculeName: RNA polymerase III / type: protein_or_peptide / ID: 1 / Name.synonym: Pol III / Oligomeric state: Complex of 17 subunits / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: Nuclear
Molecular weightExperimental: 700 KDa / Theoretical: 700 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)

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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

Concentration0.5 mg/mL
BufferpH: 7.4
Details: 10 mM Tris-HCl pH 7.4, 150 mM Ammonium sulphate, 10 mM DTT
GridDetails: 400 mesh copper rhodium grids coated with perforated carbon film
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: Controlled environment, self-built
Method: Blot for 15 s in controlled environment chamber before plunging

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
TemperatureAverage: 95 K
Alignment procedureLegacy - Astigmatism: Astigmatism was corrected at 200,000 times magnification
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 593 / Bits/pixel: 8
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 1.6 µm / Nominal magnification: 50000
Sample stageSpecimen holder: eucentric / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

Detailsca. 100000 particles were sorted into three groups according to compositional differences. The reconstruction shows the most complete species and represents ca. 48% of the particle population.
CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 10.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER
Details: Maps were calculated from defocus groups,and added. final combined map was amplitude corrected
Number images used: 40200

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