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- EMDB-1322: Insights into transcription initiation and termination from the e... -

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Basic information

Entry
Database: EMDB / ID: EMD-1322
TitleInsights into transcription initiation and termination from the electron microscopy structure of yeast RNA polymerase III.
Map dataThree volume calculated by back projection
Sample
  • Sample: yeast RNA polymerase III
  • Protein or peptide: x 17 types
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 17.0 Å
AuthorsFernandez-Tornero C / Bottcher B / Riva M / Carles C / Steuerwald U / Ruigrok RW / Sentenac A / Muller CW / Schoehn G
CitationJournal: Mol Cell / Year: 2007
Title: Insights into transcription initiation and termination from the electron microscopy structure of yeast RNA polymerase III.
Authors: Carlos Fernández-Tornero / Bettina Böttcher / Michel Riva / Christophe Carles / Ulrich Steuerwald / Rob W H Ruigrok / André Sentenac / Christoph W Müller / Guy Schoehn /
Abstract: RNA polymerase III (RNAPIII) synthesizes tRNA, 5S RNA, U6 snRNA, and other small RNAs. The structure of yeast RNAPIII, determined at 17 A resolution by cryo-electron microscopy and single-particle ...RNA polymerase III (RNAPIII) synthesizes tRNA, 5S RNA, U6 snRNA, and other small RNAs. The structure of yeast RNAPIII, determined at 17 A resolution by cryo-electron microscopy and single-particle analysis, reveals a hand-like shape typical of RNA polymerases. Compared to RNAPII, RNAPIII is characterized by a bulkier stalk and by prominent features extending from the DNA binding cleft. We attribute the latter primarily to five RNAPIII-specific subunits, present as two distinct subcomplexes (C82/C34/C31 and C53/C37). Antibody labeling experiments localize the C82/C34/C31 subcomplex to the clamp side of the DNA binding cleft, consistent with its known role in transcription initiation. The C53/C37 subcomplex appears to be situated across the cleft, near the presumed location of downstream DNA, accounting for its role in transcription termination. Our structure rationalizes available mutagenesis and biochemical data and provides insights into RNAPIII-mediated transcription.
History
DepositionFeb 6, 2007-
Header (metadata) releaseFeb 7, 2007-
Map releaseApr 20, 2007-
UpdateMar 26, 2014-
Current statusMar 26, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02113
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.02113
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1322.gif

Downloads & links

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Map

FileDownload / File: emd_1322.map.gz / Format: CCP4 / Size: 11.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThree volume calculated by back projection
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesY (Sec.)X (Row.)Z (Col.)
1.75 Å/pix.
x 144 pix.
= 252. Å		1.75 Å/pix.
x 144 pix.
= 252. Å		1.75 Å/pix.
x 144 pix.
= 252. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.75 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 0.0239 / Movie #1: 0.02113
Minimum - Maximum-0.0608731 - 0.146325
Average (Standard dev.)0.0020486 (±0.012737)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZXY
Origin-72-72-72
Dimensions144144144
Spacing144144144
CellA=B=C: 252 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.751.751.75
M x/y/z144144144
origin x/y/z0.0000.0000.000
length x/y/z252.000252.000252.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-72-72-72
NX/NY/NZ144144144
MAP C/R/S312
start NC/NR/NS-72-72-72
NC/NR/NS144144144
D min/max/mean-0.0610.1460.002

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Supplemental data

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Sample components

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Entire : yeast RNA polymerase III

EntireName: yeast RNA polymerase III
Components
  • Sample: yeast RNA polymerase III
  • Protein or peptide: C160
  • Protein or peptide: C128
  • Protein or peptide: AC40
  • Protein or peptide: AC19
  • Protein or peptide: ABC27
  • Protein or peptide: ABC23
  • Protein or peptide: ABC14.5
  • Protein or peptide: ABC10alpha
  • Protein or peptide: ABC10beta
  • Protein or peptide: C25
  • Protein or peptide: C17
  • Protein or peptide: C53
  • Protein or peptide: C37
  • Protein or peptide: C11
  • Protein or peptide: C82
  • Protein or peptide: C34
  • Protein or peptide: C31

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Supramolecule #1000: yeast RNA polymerase III

SupramoleculeName: yeast RNA polymerase III / type: sample / ID: 1000
Details: Yeast strain YOR207C containing a C-terminal tandem affinity purification tag on subunit C128 was grown on YPD medium to late log phase
Oligomeric state: The yeast enzyme is composed of seventeen components
Number unique components: 17
Molecular weightExperimental: 700 KDa / Theoretical: 700 KDa

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Macromolecule #1: C160

MacromoleculeName: C160 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: YOR207C / synonym: Baker's yeast / Tissue: yeast / Cell: yeast
Molecular weightExperimental: 160 KDa / Theoretical: 160 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #2: C128

MacromoleculeName: C128 / type: protein_or_peptide / ID: 2 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightExperimental: 128 KDa / Theoretical: 128 KDa

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Macromolecule #3: AC40

MacromoleculeName: AC40 / type: protein_or_peptide / ID: 3 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightExperimental: 40 KDa / Theoretical: 40 KDa

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Macromolecule #4: AC19

MacromoleculeName: AC19 / type: protein_or_peptide / ID: 4 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightExperimental: 19 KDa / Theoretical: 19 KDa

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Macromolecule #5: ABC27

MacromoleculeName: ABC27 / type: protein_or_peptide / ID: 5 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightExperimental: 27 KDa / Theoretical: 27 KDa

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Macromolecule #6: ABC23

MacromoleculeName: ABC23 / type: protein_or_peptide / ID: 6 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightExperimental: 23 KDa / Theoretical: 23 KDa

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Macromolecule #7: ABC14.5

MacromoleculeName: ABC14.5 / type: protein_or_peptide / ID: 7 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightExperimental: 14.5 KDa / Theoretical: 14.5 KDa

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Macromolecule #8: ABC10alpha

MacromoleculeName: ABC10alpha / type: protein_or_peptide / ID: 8 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightExperimental: 10 KDa / Theoretical: 10 KDa

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Macromolecule #9: ABC10beta

MacromoleculeName: ABC10beta / type: protein_or_peptide / ID: 9 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightExperimental: 10 KDa / Theoretical: 10 KDa

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Macromolecule #10: C25

MacromoleculeName: C25 / type: protein_or_peptide / ID: 10 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightExperimental: 25 KDa / Theoretical: 25 KDa

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Macromolecule #11: C17

MacromoleculeName: C17 / type: protein_or_peptide / ID: 11 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightExperimental: 17 KDa / Theoretical: 17 KDa

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Macromolecule #12: C53

MacromoleculeName: C53 / type: protein_or_peptide / ID: 12 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightExperimental: 53 KDa / Theoretical: 53 KDa

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Macromolecule #13: C37

MacromoleculeName: C37 / type: protein_or_peptide / ID: 13 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightExperimental: 37 KDa / Theoretical: 37 KDa

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Macromolecule #14: C11

MacromoleculeName: C11 / type: protein_or_peptide / ID: 14 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightExperimental: 11 KDa / Theoretical: 11 KDa

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Macromolecule #15: C82

MacromoleculeName: C82 / type: protein_or_peptide / ID: 15 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightExperimental: 82 KDa / Theoretical: 82 KDa

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Macromolecule #16: C34

MacromoleculeName: C34 / type: protein_or_peptide / ID: 16 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightExperimental: 34 KDa / Theoretical: 34 KDa

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Macromolecule #17: C31

MacromoleculeName: C31 / type: protein_or_peptide / ID: 17 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightExperimental: 31 KDa / Theoretical: 31 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

Concentration1.0 mg/mL
BufferpH: 8
Details: 20 mM Tris pH 8.0, 200 mM Ammonium Sulfate, 10 mM DTT
StainingType: NEGATIVE / Details: coventional Cryo EM
GridDetails: home made holey grids
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 100 K / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: modified controlled environment vitrification
Method: Manual blot for 1 second before plunging

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Electron microscopy

MicroscopeJEOL 2010F
TemperatureAverage: 100 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 100,000
DateJan 1, 2005
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 12 / Average electron dose: 10 e/Å2 / Od range: 1 / Bits/pixel: 8
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 40000 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 1.4 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 40000
Sample stageSpecimen holder: Eucentric / Specimen holder model: OTHER

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Image processing

CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: spider imagic
Details: Projection matching started with a random conical tilt model
Number images used: 10000
Final angle assignmentDetails: 386 reprojections
Final two d classificationNumber classes: 386

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Atomic model buiding 1

SoftwareName: Situs
DetailsProtocol: Rigid Body
RefinementProtocol: RIGID BODY FIT

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