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- PDB-7kpw: Structure of the H-lobe of yeast CKM -

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Basic information

Entry
Database: PDB / ID: 7kpw
TitleStructure of the H-lobe of yeast CKM
ComponentsMediator of RNA polymerase II transcription subunit 12
KeywordsTRANSCRIPTION / Mediator / Cdk8 / Med13 / Med12 / CycC / CDK / Argonaute / RNA Polymerase II / PIWI
Function / homology
Function and homology information


positive regulation of transcription by galactose / CKM complex / mediator complex / transcription coactivator activity / negative regulation of transcription by RNA polymerase II / positive regulation of transcription by RNA polymerase II
Similarity search - Function
Mediator complex, subunit Med12 / Transcription mediator complex subunit Med12 / Transcription mediator complex subunit Med12
Similarity search - Domain/homology
Mediator of RNA polymerase II transcription subunit 12
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.9 Å
AuthorsLi, Y.C. / Chao, T.C. / Kim, H.J. / Cholko, T. / Chen, S.F. / Nakanishi, K. / Chang, C.E. / Murakami, K. / Garcia, B.A. / Boyer, T.G. / Tsai, K.L.
CitationJournal: Sci Adv / Year: 2021
Title: Structure and noncanonical Cdk8 activation mechanism within an Argonaute-containing Mediator kinase module.
Authors: Yi-Chuan Li / Ti-Chun Chao / Hee Jong Kim / Timothy Cholko / Shin-Fu Chen / Guojie Li / Laura Snyder / Kotaro Nakanishi / Chia-En Chang / Kenji Murakami / Benjamin A Garcia / Thomas G Boyer / Kuang-Lei Tsai /
Abstract: The Cdk8 kinase module (CKM) in Mediator, comprising Med13, Med12, CycC, and Cdk8, regulates RNA polymerase II transcription through kinase-dependent and -independent functions. Numerous pathogenic ...The Cdk8 kinase module (CKM) in Mediator, comprising Med13, Med12, CycC, and Cdk8, regulates RNA polymerase II transcription through kinase-dependent and -independent functions. Numerous pathogenic mutations causative for neurodevelopmental disorders and cancer congregate in CKM subunits. However, the structure of the intact CKM and the mechanism by which Cdk8 is non-canonically activated and functionally affected by oncogenic CKM alterations are poorly understood. Here, we report a cryo-electron microscopy structure of CKM that redefines prior CKM structural models and explains the mechanism of Med12-dependent Cdk8 activation. Med12 interacts extensively with CycC and activates Cdk8 by stabilizing its activation (T-)loop through conserved Med12 residues recurrently mutated in human tumors. Unexpectedly, Med13 has a characteristic Argonaute-like bi-lobal architecture. These findings not only provide a structural basis for understanding CKM function and pathological dysfunction, but also further impute a previously unknown regulatory mechanism of Mediator in transcriptional modulation through its Med13 Argonaute-like features.
History
DepositionNov 12, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 27, 2021Provider: repository / Type: Initial release
Revision 1.1Feb 17, 2021Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
C: Mediator of RNA polymerase II transcription subunit 12


Theoretical massNumber of molelcules
Total (without water)167,0501
Polymers167,0501
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Mediator of RNA polymerase II transcription subunit 12 / Mediator complex subunit 12 / Suppressor of RNA polymerase B 8


Mass: 167049.812 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: SRB8, GIG1, MED12, NUT6, SSN5, YCR081W, YCR81W/YCR80W / Production host: Saccharomyces cerevisiae S288C (yeast) / References: UniProt: P25648

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: yeast CDK8 complexCyclin-dependent kinase 8 / Type: COMPLEX / Entity ID: all / Source: NATURAL
Molecular weightValue: 0.43 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Buffer solutionpH: 7.4 / Details: 25 mM Hepes pH 7.4, 200 mM NaCl, and 0.005% NP-40
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 65 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of real images: 15075
Image scansMovie frames/image: 40

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Processing

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
EM software
IDNameCategory
2EPUimage acquisition
4RELIONCTF correction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 815542
3D reconstructionResolution: 4.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 36691 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0084323
ELECTRON MICROSCOPYf_angle_d1.4326049
ELECTRON MICROSCOPYf_dihedral_angle_d1.8692593
ELECTRON MICROSCOPYf_chiral_restr0.087865
ELECTRON MICROSCOPYf_plane_restr0.007863

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