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- EMDB-8480: Cryo-EM map of the Mediator-RNAPII complex -

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Entry
Database: EMDB / ID: 8480
TitleCryo-EM map of the Mediator-RNAPII complex
Map dataMediator-RNAPII complex
Samplecomplex of Mediator and RNAPII
  • (Mediator complex subunit ...Mediator (coactivator)) x 16
  • (RNA polymerase II subunit ...) x 12
Function/homologyRNA Polymerase I Transcription Initiation / RNA Polymerase II Promoter Escape / RNA Polymerase II Transcription Initiation / Mediator complex, subunit Med27 / RNA Polymerase II Transcription Initiation And Promoter Clearance / RNA Polymerase II Transcription Pre-Initiation And Promoter Opening / mRNA Capping / Mediator complex subunit 27 / RNA Pol II CTD phosphorylation and interaction with CE / regulation of cell separation after cytokinesis ...RNA Polymerase I Transcription Initiation / RNA Polymerase II Promoter Escape / RNA Polymerase II Transcription Initiation / Mediator complex, subunit Med27 / RNA Polymerase II Transcription Initiation And Promoter Clearance / RNA Polymerase II Transcription Pre-Initiation And Promoter Opening / mRNA Capping / Mediator complex subunit 27 / RNA Pol II CTD phosphorylation and interaction with CE / regulation of cell separation after cytokinesis / Formation of the Early Elongation Complex / telomeric repeat-containing RNA transcription by RNA polymerase II / Mediator complex, subunit Med14 / TP53 Regulates Transcription of DNA Repair Genes / RNA Polymerase II Pre-transcription Events / Mediator complex, subunit Med21 / Mediator complex, subunit Med31 / Mediator complex subunit MED14 / Mediator complex, subunit Med7 / Mediator complex, subunit Med7/Med21-like / mRNA Splicing - Minor Pathway / Formation of TC-NER Pre-Incision Complex / SOH1 / Mediator complex, subunit Med6 / Mediator complex, subunit Med17 / Mediator complex, subunit Med6, fungi / Subunit 21 of Mediator complex / Mediator complex, subunit Med20 / Gap-filling DNA repair synthesis and ligation in TC-NER / MED7 protein / Dual incision in TC-NER / Mediator complex, subunit Med8, fungi/metazoa / Mediator complex, subunit Med8, fungi / Mediator of RNA polymerase II transcription subunit 22 / Mediator complex, subunit Med18 / chromatin silencing by small RNA / RNA polymerase II transcription coactivator activity involved in preinitiation complex assembly / Subunit 17 of Mediator complex / TATA-binding related factor (TRF) of subunit 20 of Mediator complex / MED6 mediator sub complex component / chromosome, centromeric outer repeat region / chromatin silencing at centromere outer repeat region / Surfeit locus protein 5 subunit 22 of Mediator complex / Mediator of RNA polymerase II transcription complex subunit 8 / Med18 protein / core mediator complex / nuclear pericentric heterochromatin / DNA-directed RNA polymerase II, holoenzyme / mediator complex / nuclear-transcribed mRNA catabolic process, exonucleolytic / RNA polymerase II, Rpb4, core / recruitment of 3'-end processing factors to RNA polymerase II holoenzyme complex / RNA polymerase II transcriptional preinitiation complex assembly / DNA-templated transcription, elongation / mRNA export from nucleus in response to heat stress / RNA polymerase II transcription cofactor activity / RNA polymerase II, Rpb4 / maintenance of transcriptional fidelity during DNA-templated transcription elongation from RNA polymerase II promoter / RNA polymerase RBP11 / Pol II subunit B9, C-terminal zinc ribbon / RNA polymerase Rpb7, N-terminal / RNA polymerase Rpb7-like, N-terminal domain superfamily / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1, domain 7 / Eukaryotic RNA polymerase II heptapeptide repeat. / RNA polymerase II, heptapeptide repeat, eukaryotic / positive regulation of translational initiation / positive regulation of nuclear-transcribed mRNA poly(A) tail shortening / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 4 / nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay / HRDC-like superfamily / Zinc finger TFIIS-type signature. / DNA-directed RNA polymerase M, 15kDa subunit, conserved site / RNA polymerases M / 15 Kd subunits signature. / DNA-directed RNA polymerase RPB5 subunit, eukaryote/virus / DNA-directed RNA polymerase subunit/transcription factor S / RNA polymerase, Rpb5, N-terminal / RNA polymerase Rpb5, N-terminal domain superfamily / DNA-directed RNA polymerase, M/15kDa subunit / DNA-directed RNA polymerase, subunit RPB6 / RNA polymerase, Rpb8 / RNA polymerase archaeal subunit P/eukaryotic subunit RPABC4 / RNA polymerases N / 8 Kd subunits signature. / RNA polymerases, subunit N, zinc binding site / RNA polymerases K / 14 to 18 Kd subunits signature. / DNA-directed RNA polymerase, subunit N/Rpb10 / Zinc finger, TFIIS-type / DNA-directed RNA polymerase, 14-18kDa subunit, conserved site / Archaeal RpoK/eukaryotic RPB6 RNA polymerase subunit / RNA polymerase subunit RPB10 / Zinc finger TFIIS-type profile. / DNA-directed RNA polymerase, 30-40kDa subunit, conserved site / RNA polymerases D / 30 to 40 Kd subunits signature. / transcription by RNA polymerase I / RPB5-like RNA polymerase subunit / RNA polymerase, subunit H/Rpb5, conserved site / Archaeal RpoH /eukaryotic RPB5 RNA polymerase subunit / RNA polymerases H / 23 Kd subunits signature. / DNA-directed RNA polymerase, RBP11-like dimerisation domain
Function and homology information
SourceSchizosaccharomyces pombe / / yeast /
MethodCryo EM / single particle reconstruction / 7.8 Å resolution
AuthorsTsai K-L / Yu X
CitationJournal: Nature / Year: 2017
Title: Mediator structure and rearrangements required for holoenzyme formation.
Authors: Kuang-Lei Tsai / Xiaodi Yu / Sneha Gopalan / Ti-Chun Chao / Ying Zhang / Laurence Florens / Michael P Washburn / Kenji Murakami / Ronald C Conaway / Joan W Conaway / Francisco J Asturias
Abstract: The conserved Mediator co-activator complex has an essential role in the regulation of RNA polymerase II transcription in all eukaryotes. Understanding the structure and interactions of Mediator is ...The conserved Mediator co-activator complex has an essential role in the regulation of RNA polymerase II transcription in all eukaryotes. Understanding the structure and interactions of Mediator is crucial for determining how the complex influences transcription initiation and conveys regulatory information to the basal transcription machinery. Here we present a 4.4 Å resolution cryo-electron microscopy map of Schizosaccharomyces pombe Mediator in which conserved Mediator subunits are individually resolved. The essential Med14 subunit works as a central backbone that connects the Mediator head, middle and tail modules. Comparison with a 7.8 Å resolution cryo-electron microscopy map of a Mediator-RNA polymerase II holoenzyme reveals that changes in the structure of Med14 facilitate a large-scale Mediator rearrangement that is essential for holoenzyme formation. Our study suggests that access to different conformations and crosstalk between structural elements are essential for the Mediator regulation mechanism, and could explain the capacity of the complex to integrate multiple regulatory signals.
Validation ReportPDB-ID: 5u0s

SummaryFull reportAbout validation report
DateDeposition: Nov 26, 2016 / Header (metadata) release: Mar 8, 2017 / Map release: Mar 8, 2017 / Last update: Sep 13, 2017

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.08
  • Imaged by UCSF CHIMERA
  • Download
  • Surface view colored by radius
  • Surface level: 0.08
  • Imaged by UCSF CHIMERA
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  • Surface view with fitted model
  • Atomic models: : PDB-5u0s
  • Surface level: 0.08
  • Imaged by UCSF CHIMERA
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-5u0s
  • Imaged by Jmol
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3D viewer
Supplemental images

Downloads & links

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Map

Fileemd_8480.map.gz (map file in CCP4 format, 21808 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
176 pix
2.62 Å/pix.
= 461.12 Å
176 pix
2.62 Å/pix.
= 461.12 Å
176 pix
2.62 Å/pix.
= 461.12 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider package.

Voxel sizeX=Y=Z: 2.62 Å
Density
Contour Level:0.08 (by emdb), 0.08 (movie #1):
Minimum - Maximum-0.11419473 - 0.22855034
Average (Standard dev.)-0.000103825936 (0.014926006)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions176176176
Origin000
Limit175175175
Spacing176176176
CellA=B=C: 461.12 Å
α=β=γ: 90 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.622.622.62
M x/y/z176176176
origin x/y/z0.0000.0000.000
length x/y/z461.120461.120461.120
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS176176176
D min/max/mean-0.1140.229-0.000

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Supplemental data

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Sample components

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Entire complex of Mediator and RNAPII

EntireName: complex of Mediator and RNAPII / Number of components: 29
MassTheoretical: 1.5 MDa

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Component #1: protein, complex of Mediator and RNAPII

ProteinName: complex of Mediator and RNAPII / Recombinant expression: No
MassTheoretical: 1.5 MDa
SourceSpecies: Schizosaccharomyces pombe

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Component #2: protein, Mediator complex subunit 6

ProteinName: Mediator complex subunit 6Mediator (coactivator) / Recombinant expression: No
MassTheoretical: 24.928947 kDa
SourceSpecies: Schizosaccharomyces pombe

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Component #3: protein, Mediator complex subunit 8

ProteinName: Mediator complex subunit 8Mediator (coactivator) / Recombinant expression: No
MassTheoretical: 23.360068 kDa
SourceSpecies: Schizosaccharomyces pombe

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Component #4: protein, Mediator complex subunit 17

ProteinName: Mediator complex subunit 17Mediator (coactivator) / Recombinant expression: No
MassTheoretical: 62.55141 kDa
SourceSpecies: Schizosaccharomyces pombe

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Component #5: protein, Mediator complex subunit 18

ProteinName: Mediator complex subunit 18Mediator (coactivator) / Recombinant expression: No
MassTheoretical: 24.056377 kDa
SourceSpecies: Schizosaccharomyces pombe

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Component #6: protein, Mediator complex subunit 20

ProteinName: Mediator complex subunit 20Mediator (coactivator) / Recombinant expression: No
MassTheoretical: 22.374775 kDa
SourceSpecies: Schizosaccharomyces pombe

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Component #7: protein, Mediator complex subunit 11

ProteinName: Mediator complex subunit 11Mediator (coactivator) / Recombinant expression: No
MassTheoretical: 13.1188 kDa
SourceSpecies: Schizosaccharomyces pombe

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Component #8: protein, Mediator complex subunit 22

ProteinName: Mediator complex subunit 22Mediator (coactivator) / Recombinant expression: No
MassTheoretical: 15.396165 kDa
SourceSpecies: Schizosaccharomyces pombe

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Component #9: protein, Mediator complex subunit 14

ProteinName: Mediator complex subunit 14Mediator (coactivator) / Recombinant expression: No
MassTheoretical: 105.382461 kDa
SourceSpecies: Schizosaccharomyces pombe

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Component #10: protein, Mediator complex subunit 4

ProteinName: Mediator complex subunit 4Mediator (coactivator) / Recombinant expression: No
MassTheoretical: 20.35801 kDa
SourceSpecies: Schizosaccharomyces pombe

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Component #11: protein, Mediator complex subunit 7

ProteinName: Mediator complex subunit 7Mediator (coactivator) / Recombinant expression: No
MassTheoretical: 43.573094 kDa
SourceSpecies: Schizosaccharomyces pombe

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Component #12: protein, Mediator complex subunit 21

ProteinName: Mediator complex subunit 21Mediator (coactivator) / Recombinant expression: No
MassTheoretical: 15.821031 kDa
SourceSpecies: Schizosaccharomyces pombe

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Component #13: protein, Mediator complex subunit 31

ProteinName: Mediator complex subunit 31Mediator (coactivator) / Recombinant expression: No
MassTheoretical: 16.913975 kDa
SourceSpecies: Schizosaccharomyces pombe

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Component #14: protein, Mediator complex subunit 27

ProteinName: Mediator complex subunit 27Mediator (coactivator) / Recombinant expression: No
MassTheoretical: 31.132693 kDa
SourceSpecies: Schizosaccharomyces pombe

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Component #15: protein, Mediator complex subunit 9

ProteinName: Mediator complex subunit 9Mediator (coactivator) / Recombinant expression: No
MassTheoretical: 13.810964 kDa
SourceSpecies: Schizosaccharomyces pombe

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Component #16: protein, Mediator complex subunit 19

ProteinName: Mediator complex subunit 19Mediator (coactivator) / Recombinant expression: No
MassTheoretical: 11.847596 kDa
SourceSpecies: Schizosaccharomyces pombe

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Component #17: protein, Mediator complex subunit 10

ProteinName: Mediator complex subunit 10Mediator (coactivator) / Recombinant expression: No
MassTheoretical: 11.251861 kDa
SourceSpecies: Schizosaccharomyces pombe

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Component #18: protein, RNA polymerase II subunit Rpb1

ProteinName: RNA polymerase II subunit Rpb1 / Recombinant expression: No
MassTheoretical: 194.372625 kDa
SourceSpecies: Schizosaccharomyces pombe

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Component #19: protein, RNA polymerase II subunit Rpb2

ProteinName: RNA polymerase II subunit Rpb2 / Recombinant expression: No
MassTheoretical: 138.027188 kDa
SourceSpecies: Schizosaccharomyces pombe

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Component #20: protein, RNA polymerase II subunit Rpb3

ProteinName: RNA polymerase II subunit Rpb3 / Recombinant expression: No
MassTheoretical: 33.748816 kDa
SourceSpecies: Schizosaccharomyces pombe

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Component #21: protein, RNA polymerase II subunit Rpb4

ProteinName: RNA polymerase II subunit Rpb4 / Recombinant expression: No
MassTheoretical: 15.37949 kDa
SourceSpecies: Schizosaccharomyces pombe

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Component #22: protein, RNA polymerase II subunit Rpb5

ProteinName: RNA polymerase II subunit Rpb5 / Recombinant expression: No
MassTheoretical: 23.954504 kDa
SourceSpecies: Schizosaccharomyces pombe

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Component #23: protein, RNA polymerase II subunit Rpb6

ProteinName: RNA polymerase II subunit Rpb6 / Recombinant expression: No
MassTheoretical: 15.742497 kDa
SourceSpecies: Schizosaccharomyces pombe

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Component #24: protein, RNA polymerase II subunit Rpb7

ProteinName: RNA polymerase II subunit Rpb7 / Recombinant expression: No
MassTheoretical: 19.121982 kDa
SourceSpecies: Schizosaccharomyces pombe

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Component #25: protein, RNA polymerase II subunit Rpb8

ProteinName: RNA polymerase II subunit Rpb8 / Recombinant expression: No
MassTheoretical: 14.317318 kDa
SourceSpecies: Schizosaccharomyces pombe

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Component #26: protein, RNA polymerase II subunit Rpb9

ProteinName: RNA polymerase II subunit Rpb9 / Recombinant expression: No
MassTheoretical: 13.192771 kDa
SourceSpecies: Schizosaccharomyces pombe

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Component #27: protein, RNA polymerase II subunit Rpb10

ProteinName: RNA polymerase II subunit Rpb10 / Recombinant expression: No
MassTheoretical: 8.286801 kDa
SourceSpecies: Schizosaccharomyces pombe

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Component #28: protein, RNA polymerase II subunit Rpb11

ProteinName: RNA polymerase II subunit Rpb11 / Recombinant expression: No
MassTheoretical: 14.143276 kDa
SourceSpecies: Schizosaccharomyces pombe

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Component #29: protein, RNA polymerase II subunit Rpb12

ProteinName: RNA polymerase II subunit Rpb12 / Recombinant expression: No
MassTheoretical: 7.216495 kDa
SourceSpecies: Schizosaccharomyces pombe

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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: 20 mM HEPES, pH 7.4, 200 mM potassium acetate, 5 mM b-Mercaptoethanol, 0.01% NP-40
pH: 7.4
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 9.6 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 22500 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000 - 4000 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 3862
3D reconstructionSoftware: RELION / Resolution: 7.8 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution assessment)

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Atomic model buiding

Output model

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