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Yorodumi- EMDB-5589: Electron cryo-microscopy of the yeast Mediator Cdk8 kinase module -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-5589 | |||||||||
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Title | Electron cryo-microscopy of the yeast Mediator Cdk8 kinase module | |||||||||
Map data | A map of a complex formed by yeast Mediator and Cdk8 kinase module | |||||||||
Sample |
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Keywords | Mediator / kinase module / CDK8 / Med12 / Med13 / CycC | |||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 35.0 Å | |||||||||
Authors | Tsai KL / Sato S / Tomomori-Sato C / Conaway RC / Conaway JW / Asturias FJ | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
-Downloads & links
-EMDB archive
Map data | emd_5589.map.gz | 1.5 MB | EMDB map data format | |
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Header (meta data) | emd-5589-v30.xml emd-5589.xml | 10.2 KB 10.2 KB | Display Display | EMDB header |
Images | emd_5589.jpg | 78.8 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-5589 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-5589 | HTTPS FTP |
-Validation report
Summary document | emd_5589_validation.pdf.gz | 78.5 KB | Display | EMDB validaton report |
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Full document | emd_5589_full_validation.pdf.gz | 77.6 KB | Display | |
Data in XML | emd_5589_validation.xml.gz | 494 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5589 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5589 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_5589.map.gz / Format: CCP4 / Size: 1.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | A map of a complex formed by yeast Mediator and Cdk8 kinase module | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 8.7 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : A complex between the yeast Mediator and Cdk8 kinase module complexes
Entire | Name: A complex between the yeast Mediator and Cdk8 kinase module complexes |
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Components |
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-Supramolecule #1000: A complex between the yeast Mediator and Cdk8 kinase module complexes
Supramolecule | Name: 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 |
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Molecular weight | Experimental: 1.5 MDa / Theoretical: 1.5 MDa |
-Macromolecule #1: Mediator complex
Macromolecule | Name: Mediator complex / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: yeast |
Molecular weight | Experimental: 1.1 MDa / Theoretical: 1.1 MDa |
-Macromolecule #2: Cdk8 kinase module
Macromolecule | Name: Cdk8 kinase module / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: yeast |
Molecular weight | Experimental: 400 KDa / Theoretical: 400 KDa |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.020 mg/mL |
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Buffer | pH: 7.6 Details: 20mM HEPES, 200mM potassium acetate, 5mM beta-mercaptoethanol |
Staining | Type: 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 |
Grid | Details: 300 mesh Cu/Rh grids with continuous carbon, glow discharged in amylamine atmosphere |
Vitrification | Cryogen name: NONE / Instrument: OTHER |
-Electron microscopy
Microscope | FEI TECNAI F20 |
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Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 125,000 times magnification using a CCD camera |
Date | Jun 1, 2012 |
Image recording | Category: CCD / Film or detector model: GENERIC CCD / Number real images: 200 / Average electron dose: 20 e/Å2 |
Electron beam | Acceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 1.2 mm / Nominal defocus max: 0.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder model: SIDE ENTRY, EUCENTRIC |
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company |
-Image processing
Final reconstruction | Algorithm: 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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