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- EMDB-22989: Structure of kinase and Central lobes of yeast CKM -

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Basic information

Entry
Database: EMDB / ID: EMD-22989
TitleStructure of kinase and Central lobes of yeast CKM
Map data
Sampleyeast CDK8 complexCyclin-dependent kinase 8
  • Meiotic mRNA stability protein kinase SSN3
  • RNA polymerase II holoenzyme cyclin-like subunit
  • (Mediator of RNA polymerase II transcription subunit ...) x 2
Function / homology
Function and homology information


negative regulation of filamentous growth / positive regulation of transcription from RNA polymerase II promoter involved in meiotic cell cycle / positive regulation of transcription by galactose / nuclear-transcribed mRNA catabolic process, non-stop decay / [RNA-polymerase]-subunit kinase / phosphorylation of RNA polymerase II C-terminal domain / mediator complex / cyclin-dependent protein serine/threonine kinase regulator activity / positive regulation of transcription from RNA polymerase II promoter by galactose / RNA polymerase II core promoter sequence-specific DNA binding ...negative regulation of filamentous growth / positive regulation of transcription from RNA polymerase II promoter involved in meiotic cell cycle / positive regulation of transcription by galactose / nuclear-transcribed mRNA catabolic process, non-stop decay / [RNA-polymerase]-subunit kinase / phosphorylation of RNA polymerase II C-terminal domain / mediator complex / cyclin-dependent protein serine/threonine kinase regulator activity / positive regulation of transcription from RNA polymerase II promoter by galactose / RNA polymerase II core promoter sequence-specific DNA binding / cyclin-dependent kinase / cyclin-dependent protein serine/threonine kinase activity / RNA polymerase II CTD heptapeptide repeat kinase activity / meiotic cell cycle / protein destabilization / transcription coregulator activity / transcription coactivator activity / response to oxidative stress / protein kinase activity / regulation of transcription by RNA polymerase II / protein phosphorylation / negative regulation of transcription by RNA polymerase II / positive regulation of transcription by RNA polymerase II / ATP binding / metal ion binding / nucleus
Mediator complex, subunit Med12 / Mediator complex, subunit Med13, N-terminal / Cyclin-like / Protein kinase-like domain superfamily / Mediator complex subunit Med13, C-terminal / Serine/threonine-protein kinase, active site / Cyclin, N-terminal / Protein kinase domain / Cyclin C/cyclin-like subunit Ssn8 / Cyclin/Cyclin-like subunit Ssn8 / Cyclin-like superfamily
Mediator of RNA polymerase II transcription subunit 12 / Mediator of RNA polymerase II transcription subunit 13 / Meiotic mRNA stability protein kinase SSN3 / RNA polymerase II holoenzyme cyclin-like subunit
Biological speciesSaccharomyces cerevisiae S288C (yeast) / Baker's yeast (baker's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsLi YC / Chao TC / Kim HJ / Cholko T / Chen SF / Nakanishi K / Chang CE / Murakami K / Garcia BA / Boyer TG / Tsai KL
Funding support United States, 1 items
OrganizationGrant numberCountry
Cancer Prevention and Research Institute of Texas (CPRIT)13127 United States
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.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionNov 12, 2020-
Header (metadata) releaseJan 27, 2021-
Map releaseJan 27, 2021-
UpdateFeb 17, 2021-
Current statusFeb 17, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.029
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.029
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7kpv
  • Surface level: 0.022
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_22989.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 384 pix.
= 410.88 Å
1.07 Å/pix.
x 384 pix.
= 410.88 Å
1.07 Å/pix.
x 384 pix.
= 410.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.029 / Movie #1: 0.029
Minimum - Maximum-0.058909096 - 0.1168836
Average (Standard dev.)8.147647e-05 (±0.0030819105)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 410.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z410.880410.880410.880
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.0590.1170.000

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Supplemental data

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Half map: half-volume 1

Fileemd_22989_half_map_1.map
Annotationhalf-volume 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: half-volume 2

Fileemd_22989_half_map_2.map
Annotationhalf-volume 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire yeast CDK8 complex

EntireName: yeast CDK8 complexCyclin-dependent kinase 8 / Number of components: 5

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Component #1: protein, yeast CDK8 complex

ProteinName: yeast CDK8 complexCyclin-dependent kinase 8 / Recombinant expression: No
MassTheoretical: 430 kDa
SourceSpecies: Saccharomyces cerevisiae S288C (yeast)

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Component #2: protein, Meiotic mRNA stability protein kinase SSN3

ProteinName: Meiotic mRNA stability protein kinase SSN3 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 62.940906 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: ATCC 204508 / S288c

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Component #3: protein, RNA polymerase II holoenzyme cyclin-like subunit

ProteinName: RNA polymerase II holoenzyme cyclin-like subunit / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 37.834598 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: ATCC 204508 / S288c

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Component #4: protein, Mediator of RNA polymerase II transcription subunit 12

ProteinName: Mediator of RNA polymerase II transcription subunit 12
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 167.049812 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: ATCC 204508 / S288c

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Component #5: protein, Mediator of RNA polymerase II transcription subunit 13

ProteinName: Mediator of RNA polymerase II transcription subunit 13
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 160.175156 kDa
SourceSpecies: Baker's yeast (baker's yeast) / Strain: ATCC 204508 / S288c

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 1 mg/mL
Buffer solution: 25 mM Hepes pH 7.4, 200 mM NaCl, and 0.005% NP-40
pH: 7.4
Support filmunspecified
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
ImagingMicroscope: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 65 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 QUANTUM (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 31534
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Output model

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