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Yorodumi- EMDB-5588: Electron cryo-microscopy of the yeast Mediator Cdk8 kinase module -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-5588 | |||||||||
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Title | Electron cryo-microscopy of the yeast Mediator Cdk8 kinase module | |||||||||
Map data | Map of the CDK8 kinase module including Med12, Med13, Cdk8, and CycC | |||||||||
Sample |
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Keywords | Mediator / kinase module / CDK8 / Med12 / Med13 / CycC | |||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 15.0 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_5588.map.gz | 10.5 MB | EMDB map data format | |
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Header (meta data) | emd-5588-v30.xml emd-5588.xml | 12.2 KB 12.2 KB | Display Display | EMDB header |
Images | emd_5588.jpg | 71.5 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-5588 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-5588 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_5588.map.gz / Format: CCP4 / Size: 11.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Map of the CDK8 kinase module including Med12, Med13, Cdk8, and CycC | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.1 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 : Cdk8 kinase module of yeast Mediator complex
Entire | Name: Cdk8 kinase module of yeast Mediator complex |
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Components |
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-Supramolecule #1000: Cdk8 kinase module of yeast Mediator complex
Supramolecule | Name: Cdk8 kinase module of yeast Mediator complex / type: sample / ID: 1000 / Number unique components: 4 |
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Molecular weight | Experimental: 430 KDa / Theoretical: 430 KDa |
-Macromolecule #1: Med12
Macromolecule | Name: Med12 / type: protein_or_peptide / ID: 1 / Name.synonym: Srb8 / 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: 167 KDa / Theoretical: 167 KDa |
-Macromolecule #2: Med13
Macromolecule | Name: Med13 / type: protein_or_peptide / ID: 2 / Name.synonym: Srb9 / 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: 160 KDa / Theoretical: 160 KDa |
-Macromolecule #3: Cdk8
Macromolecule | Name: Cdk8 / type: protein_or_peptide / ID: 3 / Name.synonym: Srb10 / 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: 63 KDa / Theoretical: 63 KDa |
-Macromolecule #4: CycC
Macromolecule | Name: CycC / type: protein_or_peptide / ID: 4 / Name.synonym: Srb11 / 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: 38 KDa / Theoretical: 38 KDa |
-Experimental details
-Structure determination
Method | cryo EM |
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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 |
Grid | Details: 300 mesh Cu/Rh grids with continuous carbon, glow discharged in amylamine atmosphere |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 77 K / Instrument: HOMEMADE PLUNGER |
-Electron microscopy
Microscope | FEI TECNAI F20 |
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Electron beam | Acceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 60400 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 1.2 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 60000 |
Sample stage | Specimen holder model: GATAN LIQUID NITROGEN |
Temperature | Min: 80 K / Max: 110 K / Average: 105 K |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 125,000 times magnification using a CCD camera |
Date | Dec 1, 2011 |
Image recording | Category: 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 |
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company |
-Image processing
CTF correction | Details: Each particle |
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Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 15.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPARX, SPIDER / Number images used: 70000 |