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- EMDB-5589: Electron cryo-microscopy of the yeast Mediator Cdk8 kinase module -

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Basic information

Entry
Database: EMDB / ID: EMD-5589
TitleElectron cryo-microscopy of the yeast Mediator Cdk8 kinase module
Map dataA map of a complex formed by yeast Mediator and Cdk8 kinase module
Sample
  • Sample: A complex between the yeast Mediator and Cdk8 kinase module complexes
  • Protein or peptide: Mediator complexMediator (coactivator)
  • Protein or peptide: Cdk8 kinase module
KeywordsMediator / kinase module / CDK8 / Med12 / Med13 / CycC
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 35.0 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: 1.4
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1.4
  • 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_5589.map.gz / Format: CCP4 / Size: 1.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationA map of a complex formed by yeast Mediator and Cdk8 kinase module
Voxel sizeX=Y=Z: 8.7 Å
Density
Contour LevelBy AUTHOR: 1.25 / Movie #1: 1.4
Minimum - Maximum-3.22206473 - 5.8071847
Average (Standard dev.)0.00549408 (±0.35821825)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-36-36-36
Dimensions747474
Spacing747474
CellA=B=C: 643.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z8.78.78.7
M x/y/z747474
origin x/y/z0.0000.0000.000
length x/y/z643.800643.800643.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-5029166
NX/NY/NZ106122134
MAP C/R/S123
start NC/NR/NS-36-36-36
NC/NR/NS747474
D min/max/mean-3.2225.8070.005

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Supplemental data

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Sample components

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Entire : A complex between the yeast Mediator and Cdk8 kinase module complexes

EntireName: A complex between the yeast Mediator and Cdk8 kinase module complexes
Components
  • Sample: A complex between the yeast Mediator and Cdk8 kinase module complexes
  • Protein or peptide: Mediator complexMediator (coactivator)
  • Protein or peptide: Cdk8 kinase module

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Supramolecule #1000: A complex between the yeast Mediator and Cdk8 kinase module complexes

SupramoleculeName: A complex between the yeast Mediator and Cdk8 kinase module complexes
type: sample / ID: 1000
Details: The complex was formed by incubating purified Mediator and Cdk8 kinase module
Number unique components: 2
Molecular weightExperimental: 1.5 MDa / Theoretical: 1.5 MDa

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Macromolecule #1: Mediator complex

MacromoleculeName: Mediator complex / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: yeast
Molecular weightExperimental: 1.1 MDa / Theoretical: 1.1 MDa

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Macromolecule #2: Cdk8 kinase module

MacromoleculeName: Cdk8 kinase module / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: yeast
Molecular weightExperimental: 400 KDa / Theoretical: 400 KDa

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Experimental details

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Structure determination

Methodnegative staining
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
StainingType: NEGATIVE
Details: Grids with adsorbed protein were stained with a drop of 2% uranyl acetate for about 30 sec and then covered with a second thin carbon layer
GridDetails: 300 mesh Cu/Rh grids with continuous carbon, glow discharged in amylamine atmosphere
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 1.2 mm / Nominal defocus max: 0.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 125,000 times magnification using a CCD camera
DateJun 1, 2012
Image recordingCategory: CCD / Film or detector model: GENERIC CCD / Number real images: 200 / Average electron dose: 20 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 35.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPARX, SPIDER / Number images used: 2000

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