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- PDB-5u0p: Cryo-EM structure of the transcriptional Mediator -

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Basic information

Entry
Database: PDB / ID: 5u0p
TitleCryo-EM structure of the transcriptional Mediator
Components(Mediator complex subunit ...Mediator (coactivator)) x 16
KeywordsTRANSCRIPTION / transcriptional / Mediator / Head / Middle / Tail / Srb / RNA polymerase II / Med
Function / homology
Function and homology information


core mediator complex / mediator complex / termination of RNA polymerase II transcription / positive regulation of transcription initiation by RNA polymerase II / transcription initiation at RNA polymerase II promoter / transcription coregulator activity / euchromatin / transcription coactivator activity / RNA polymerase II cis-regulatory region sequence-specific DNA binding / regulation of transcription by RNA polymerase II ...core mediator complex / mediator complex / termination of RNA polymerase II transcription / positive regulation of transcription initiation by RNA polymerase II / transcription initiation at RNA polymerase II promoter / transcription coregulator activity / euchromatin / transcription coactivator activity / RNA polymerase II cis-regulatory region sequence-specific DNA binding / regulation of transcription by RNA polymerase II / nucleus / cytoplasm
Similarity search - Function
Mediator complex, subunit Med27 / Mediator complex subunit 27 / Mediator complex, subunit Med6, fungi / Mediator complex, subunit Med7 superfmaily / Mediator complex, subunit Med31 / Mediator complex, subunit Med21 / Mediator of RNA polymerase II, subunit Med31 superfamily / SOH1 / Subunit 21 of Mediator complex / Mediator complex, subunit Med7 ...Mediator complex, subunit Med27 / Mediator complex subunit 27 / Mediator complex, subunit Med6, fungi / Mediator complex, subunit Med7 superfmaily / Mediator complex, subunit Med31 / Mediator complex, subunit Med21 / Mediator of RNA polymerase II, subunit Med31 superfamily / SOH1 / Subunit 21 of Mediator complex / Mediator complex, subunit Med7 / Mediator complex, subunit Med7/Med21-like / MED7 protein / Mediator complex, subunit Med20 / Mediator complex, subunit Med14 / TATA-binding related factor (TRF) of subunit 20 of Mediator complex / Mediator complex subunit MED14 / Mediator complex, subunit Med6 / Mediator complex, subunit Med17 / Mediator complex, subunit Med6 superfamily / MED6 mediator sub complex component / Subunit 17 of Mediator complex / Mediator of RNA polymerase II transcription subunit 22 / Mediator complex, subunit Med8, fungi/metazoa / Mediator complex, subunit Med11 / Surfeit locus protein 5 subunit 22 of Mediator complex / Mediator of RNA polymerase II transcription complex subunit 8 / Mediator complex protein / Mediator complex, subunit Med18 / Med18 protein
Similarity search - Domain/homology
Uncharacterized protein C24C9.04 / Mediator of RNA polymerase II transcription subunit 22 / Mediator of RNA polymerase II transcription subunit 18 / Mediator of RNA polymerase II transcription subunit 7 / Mediator of RNA polymerase II transcription subunit 21 / Mediator of RNA polymerase II transcription subunit 8 / Mediator of RNA polymerase II transcription subunit 17 / Mediator of RNA polymerase II transcription subunit 20 / Mediator of RNA polymerase II transcription subunit 27 / Mediator of RNA polymerase II transcription subunit 11 ...Uncharacterized protein C24C9.04 / Mediator of RNA polymerase II transcription subunit 22 / Mediator of RNA polymerase II transcription subunit 18 / Mediator of RNA polymerase II transcription subunit 7 / Mediator of RNA polymerase II transcription subunit 21 / Mediator of RNA polymerase II transcription subunit 8 / Mediator of RNA polymerase II transcription subunit 17 / Mediator of RNA polymerase II transcription subunit 20 / Mediator of RNA polymerase II transcription subunit 27 / Mediator of RNA polymerase II transcription subunit 11 / Mediator of RNA polymerase II transcription subunit 14 / Mediator of RNA polymerase II transcription subunit 6 / Mediator of RNA polymerase II transcription subunit 31
Similarity search - Component
Biological speciesSchizosaccharomyces pombe (fission yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsTsai, K.-L. / Yu, X. / Gopalan, S. / Chao, T.-C. / Zhang, Y. / Florens, L. / Washburn, M.P. / Murakami, K. / Conaway, R.C. / Conaway, J.W. / Asturias, F.
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.
History
DepositionNov 26, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 8, 2017Provider: repository / Type: Initial release
Revision 1.1Mar 15, 2017Group: Database references
Revision 1.2Apr 19, 2017Group: Database references
Revision 1.3Jul 18, 2018Group: Data collection / Category: em_image_scans / em_software / Item: _em_software.name
Revision 1.4Mar 13, 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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Structure visualization

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Assembly

Deposited unit
N: Mediator complex subunit 14
F: Mediator complex subunit 6
H: Mediator complex subunit 8
Q: Mediator complex subunit 17
R: Mediator complex subunit 18
T: Mediator complex subunit 20
K: Mediator complex subunit 11
V: Mediator complex subunit 22
D: Mediator complex subunit 4
G: Mediator complex subunit 7
U: Mediator complex subunit 21
3: Mediator complex subunit 31
I: Mediator complex subunit 9
2: Mediator complex subunit 27
S: Mediator complex subunit 19
J: Mediator complex subunit 10


Theoretical massNumber of molelcules
Total (without water)455,87816
Polymers455,87816
Non-polymers00
Water0
1


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

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Components

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Mediator complex subunit ... , 16 types, 16 molecules NFHQRTKVDGU3I2SJ

#1: Protein Mediator complex subunit 14 / Mediator (coactivator)


Mass: 105382.461 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Schizosaccharomyces pombe (fission yeast) / References: UniProt: Q9P7Y4
#2: Protein Mediator complex subunit 6 / Mediator (coactivator)


Mass: 24928.947 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Schizosaccharomyces pombe (fission yeast) / References: UniProt: Q9US45
#3: Protein Mediator complex subunit 8 / Mediator (coactivator)


Mass: 23360.068 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Schizosaccharomyces pombe (fission yeast) / References: UniProt: O94646
#4: Protein Mediator complex subunit 17 / Mediator (coactivator)


Mass: 62551.410 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Schizosaccharomyces pombe (fission yeast) / References: UniProt: P87306
#5: Protein Mediator complex subunit 18 / Mediator (coactivator)


Mass: 24056.377 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Schizosaccharomyces pombe (fission yeast) / References: UniProt: O14198
#6: Protein Mediator complex subunit 20 / Mediator (coactivator)


Mass: 22374.775 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Schizosaccharomyces pombe (fission yeast) / References: UniProt: Q10317
#7: Protein Mediator complex subunit 11 / Mediator (coactivator)


Mass: 13118.800 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Schizosaccharomyces pombe (fission yeast) / References: UniProt: Q9P6Q0
#8: Protein Mediator complex subunit 22 / Mediator (coactivator)


Mass: 15396.165 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Schizosaccharomyces pombe (fission yeast) / References: UniProt: O14010
#9: Protein Mediator complex subunit 4 / Mediator (coactivator)


Mass: 20358.010 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Schizosaccharomyces pombe (fission yeast)
#10: Protein Mediator complex subunit 7 / Mediator (coactivator)


Mass: 43573.094 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Schizosaccharomyces pombe (fission yeast) / References: UniProt: O60104
#11: Protein Mediator complex subunit 21 / Mediator (coactivator)


Mass: 15821.031 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Schizosaccharomyces pombe (fission yeast) / References: UniProt: O94376
#12: Protein Mediator complex subunit 31 / Mediator (coactivator)


Mass: 16913.975 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Schizosaccharomyces pombe (fission yeast) / References: UniProt: Q9USH1
#13: Protein Mediator complex subunit 9 / Mediator (coactivator)


Mass: 13810.964 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Schizosaccharomyces pombe (fission yeast) / References: UniProt: O13964
#14: Protein Mediator complex subunit 27 / Mediator (coactivator)


Mass: 31132.693 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Schizosaccharomyces pombe (fission yeast) / References: UniProt: Q10477
#15: Protein Mediator complex subunit 19 / Mediator (coactivator)


Mass: 11847.596 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Schizosaccharomyces pombe (fission yeast)
#16: Protein Mediator complex subunit 10 / Mediator (coactivator)


Mass: 11251.861 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Schizosaccharomyces pombe (fission yeast)

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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: Mediator complexMediator (coactivator) / Type: COMPLEX / Entity ID: all / Source: NATURAL
Source (natural)Organism: Schizosaccharomyces pombe (fission yeast)
Buffer solutionpH: 7.4
Details: 20 mM HEPES, pH 7.4, 200 mM potassium acetate, 5 mM b-Mercaptoethanol, 0.01% NP-40
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 22500 X / Nominal defocus max: 4000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 7 sec. / Electron dose: 9.6 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 11181

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Processing

EM software
IDNameVersionCategory
1Appionparticle selection
2Leginon3image acquisition
4CTFFIND3CTF correction
10RELION1.4initial Euler assignment
11RELION1.4final Euler assignment
13RELION1.43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 42484 / Symmetry type: POINT

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