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- EMDB-5588: Electron cryo-microscopy of the yeast Mediator Cdk8 kinase module -

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Basic information

Entry
Database: EMDB / ID: EMD-5588
TitleElectron cryo-microscopy of the yeast Mediator Cdk8 kinase module
Map dataMap of the CDK8 kinase module including Med12, Med13, Cdk8, and CycC
Sample
  • Sample: Cdk8 kinase module of yeast Mediator complex
  • Protein or peptide: Med12
  • Protein or peptide: Med13
  • Protein or peptide: Cdk8
  • Protein or peptide: CycC
KeywordsMediator / kinase module / CDK8 / Med12 / Med13 / CycC
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 15.0 Å
AuthorsTsai KL / Sato S / Tomomori-Sato C / Conaway RC / Conaway JW / Asturias FJ
CitationJournal: Nat Struct Mol Biol / Year: 2013
Title: A conserved Mediator-CDK8 kinase module association regulates Mediator-RNA polymerase II interaction.
Authors: Kuang-Lei Tsai / Shigeo Sato / Chieri Tomomori-Sato / Ronald C Conaway / Joan W Conaway / Francisco J Asturias /
Abstract: The CDK8 kinase module (CKM) is a conserved, dissociable Mediator subcomplex whose component subunits were genetically linked to the RNA polymerase II (RNAPII) C-terminal domain (CTD) and ...The CDK8 kinase module (CKM) is a conserved, dissociable Mediator subcomplex whose component subunits were genetically linked to the RNA polymerase II (RNAPII) C-terminal domain (CTD) and individually recognized as transcriptional repressors before Mediator was identified as a pre-eminent complex in eukaryotic transcription regulation. We used macromolecular EM and biochemistry to investigate the subunit organization, structure and Mediator interaction of the Saccharomyces cerevisiae CKM. We found that interaction of the CKM with Mediator's middle module interferes with CTD-dependent RNAPII binding to a previously unknown middle-module CTD-binding site and with the holoenzyme formation process. Taken together, our results reveal the basis for CKM repression, clarify the origin of the connection between CKM subunits and the CTD and suggest that a combination of competitive interactions and conformational changes that facilitate holoenzyme formation underlie the mechanism of transcription regulation by Mediator.
History
DepositionFeb 26, 2013-
Header (metadata) releaseMar 20, 2013-
Map releaseApr 17, 2013-
UpdateMay 15, 2013-
Current statusMay 15, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.001
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.001
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol

Downloads & links

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Map

FileDownload / File: emd_5588.map.gz / Format: CCP4 / Size: 11.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of the CDK8 kinase module including Med12, Med13, Cdk8, and CycC
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.1 Å/pix.
x 144 pix.
= 302.4 Å		2.1 Å/pix.
x 144 pix.
= 302.4 Å		2.1 Å/pix.
x 144 pix.
= 302.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.00105 / Movie #1: 0.001
Minimum - Maximum-0.01605716 - 0.03793941
Average (Standard dev.)0.00001135 (±0.00171779)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions144144144
Spacing144144144
CellA=B=C: 302.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.12.12.1
M x/y/z144144144
origin x/y/z0.0000.0000.000
length x/y/z302.400302.400302.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-5029166
NX/NY/NZ106122134
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS144144144
D min/max/mean-0.0160.0380.000

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Supplemental data

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Sample components

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Entire : Cdk8 kinase module of yeast Mediator complex

EntireName: Cdk8 kinase module of yeast Mediator complex
Components
  • Sample: Cdk8 kinase module of yeast Mediator complex
  • Protein or peptide: Med12
  • Protein or peptide: Med13
  • Protein or peptide: Cdk8
  • Protein or peptide: CycC

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Supramolecule #1000: Cdk8 kinase module of yeast Mediator complex

SupramoleculeName: Cdk8 kinase module of yeast Mediator complex / type: sample / ID: 1000 / Number unique components: 4
Molecular weightExperimental: 430 KDa / Theoretical: 430 KDa

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Macromolecule #1: Med12

MacromoleculeName: Med12 / type: protein_or_peptide / ID: 1 / Name.synonym: Srb8 / Number of copies: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: yeast
Molecular weightExperimental: 167 KDa / Theoretical: 167 KDa

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Macromolecule #2: Med13

MacromoleculeName: Med13 / type: protein_or_peptide / ID: 2 / Name.synonym: Srb9 / Number of copies: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: yeast
Molecular weightExperimental: 160 KDa / Theoretical: 160 KDa

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Macromolecule #3: Cdk8

MacromoleculeName: Cdk8 / type: protein_or_peptide / ID: 3 / Name.synonym: Srb10 / Number of copies: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: yeast
Molecular weightExperimental: 63 KDa / Theoretical: 63 KDa

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Macromolecule #4: CycC

MacromoleculeName: CycC / type: protein_or_peptide / ID: 4 / Name.synonym: Srb11 / Number of copies: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: yeast
Molecular weightExperimental: 38 KDa / Theoretical: 38 KDa

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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

Concentration0.020 mg/mL
BufferpH: 7.6
Details: 20mM HEPES, 200mM potassium acetate, 5mM beta-mercaptoethanol
GridDetails: 300 mesh Cu/Rh grids with continuous carbon, glow discharged in amylamine atmosphere
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 77 K / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureMin: 80 K / Max: 110 K / Average: 105 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 125,000 times magnification using a CCD camera
DateDec 1, 2011
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 6.35 µm / Number real images: 154 / Average electron dose: 15 e/Å2 / Bits/pixel: 16
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 60400 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 1.2 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 60000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 15.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPARX, SPIDER / Number images used: 70000

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