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- EMDB-23259: Focused refinement of the Mediator Middle module and TFIIH CAK mo... -

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Basic information

Entry
Database: EMDB / ID: EMD-23259
TitleFocused refinement of the Mediator Middle module and TFIIH CAK module within the human Mediator-bound transcription pre-initiation complex
Map data
Sample
  • Complex: Mediator-bound pre-initiation complex
    • Complex: RNA Polymerase II
    • Complex: Mediator
    • Complex: TFIIHTranscription factor II H
    • Complex: General transcription factorsGeneral transcription factor
    • Complex: promoter DNA
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.5 Å
AuthorsAbdella R / Talyzina A / He Y
Funding support United States, 5 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
American Cancer SocietyIRG-15-173-21 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)P01-CA092584 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)U54-CA193419 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32-GM008382 United States
CitationJournal: Science / Year: 2021
Title: Structure of the human Mediator-bound transcription preinitiation complex.
Authors: R Abdella / A Talyzina / S Chen / C J Inouye / R Tjian / Y He /
Abstract: Eukaryotic transcription requires the assembly of a multisubunit preinitiation complex (PIC) composed of RNA polymerase II (Pol II) and the general transcription factors. The coactivator Mediator is ...Eukaryotic transcription requires the assembly of a multisubunit preinitiation complex (PIC) composed of RNA polymerase II (Pol II) and the general transcription factors. The coactivator Mediator is recruited by transcription factors, facilitates the assembly of the PIC, and stimulates phosphorylation of the Pol II C-terminal domain (CTD) by the TFIIH subunit CDK7. Here, we present the cryo-electron microscopy structure of the human Mediator-bound PIC at a resolution below 4 angstroms. Transcription factor binding sites within Mediator are primarily flexibly tethered to the tail module. CDK7 is stabilized by multiple contacts with Mediator. Two binding sites exist for the Pol II CTD, one between the head and middle modules of Mediator and the other in the active site of CDK7, providing structural evidence for Pol II CTD phosphorylation within the Mediator-bound PIC.
History
DepositionJan 8, 2021-
Header (metadata) releaseMar 24, 2021-
Map releaseMar 24, 2021-
UpdateApr 14, 2021-
Current statusApr 14, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23259.png

Downloads & links

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Map

FileDownload / File: emd_23259.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.059 Å
Density
Contour LevelBy AUTHOR: 0.1 / Movie #1: 0.1
Minimum - Maximum-0.0065271626 - 1.6062171
Average (Standard dev.)0.004279392 (±0.04099157)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin230210280
Dimensions240240240
Spacing240240240
CellA=B=C: 254.16 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0591.0591.059
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z254.160254.160254.160
α/β/γ90.00090.00090.000
start NX/NY/NZ10510593
NX/NY/NZ143147156
MAP C/R/S123
start NC/NR/NS210230280
NC/NR/NS240240240
D min/max/mean-0.0071.6060.004

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Supplemental data

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Sample components

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Entire : Mediator-bound pre-initiation complex

EntireName: Mediator-bound pre-initiation complex
Components
  • Complex: Mediator-bound pre-initiation complex
    • Complex: RNA Polymerase II
    • Complex: Mediator
    • Complex: TFIIHTranscription factor II H
    • Complex: General transcription factorsGeneral transcription factor
    • Complex: promoter DNA

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Supramolecule #1: Mediator-bound pre-initiation complex

SupramoleculeName: Mediator-bound pre-initiation complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#56
Molecular weightTheoretical: 2.7 MDa

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Supramolecule #2: RNA Polymerase II

SupramoleculeName: RNA Polymerase II / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#12
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 520 KDa

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Supramolecule #3: Mediator

SupramoleculeName: Mediator / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #33-#56
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 1.4 MDa

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Supramolecule #4: TFIIH

SupramoleculeName: TFIIH / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #23-#32
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 500 KDa

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Supramolecule #5: General transcription factors

SupramoleculeName: General transcription factors / type: complex / ID: 5 / Parent: 1 / Macromolecule list: #13-#20
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 300 KDa

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Supramolecule #6: promoter DNA

SupramoleculeName: promoter DNA / type: complex / ID: 6 / Parent: 1 / Macromolecule list: #21-#22
Source (natural)Organism: Homo sapiens (human)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.9
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 31.5 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 43779

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