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- EMDB-2309: Structural visualization of key steps in human transcription init... -

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Basic information

Entry
Database: EMDB / ID: EMD-2309
TitleStructural visualization of key steps in human transcription initiation
Map dataapo TFIIH
Sample
  • Sample: apo TFIIH
  • Protein or peptide: General transcription factor IIH
Keywordshuman transcription initiation
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 20.0 Å
AuthorsHe Y / Fang J / Taatjes DJ / Nogales E
CitationJournal: Nature / Year: 2013
Title: Structural visualization of key steps in human transcription initiation.
Authors: Yuan He / Jie Fang / Dylan J Taatjes / Eva Nogales /
Abstract: Eukaryotic transcription initiation requires the assembly of general transcription factors into a pre-initiation complex that ensures the accurate loading of RNA polymerase II (Pol II) at the ...Eukaryotic transcription initiation requires the assembly of general transcription factors into a pre-initiation complex that ensures the accurate loading of RNA polymerase II (Pol II) at the transcription start site. The molecular mechanism and function of this assembly have remained elusive due to lack of structural information. Here we have used an in vitro reconstituted system to study the stepwise assembly of human TBP, TFIIA, TFIIB, Pol II, TFIIF, TFIIE and TFIIH onto promoter DNA using cryo-electron microscopy. Our structural analyses provide pseudo-atomic models at various stages of transcription initiation that illuminate critical molecular interactions, including how TFIIF engages Pol II and promoter DNA to stabilize both the closed pre-initiation complex and the open-promoter complex, and to regulate start--initiation complexes, combined with the localization of the TFIIH helicases XPD and XPB, support a DNA translocation model of XPB and explain its essential role in promoter opening.
History
DepositionFeb 5, 2013-
Header (metadata) releaseMar 6, 2013-
Map releaseMar 13, 2013-
UpdateApr 10, 2013-
Current statusApr 10, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.4
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 2.4
  • 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_2309.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationapo TFIIH
Voxel sizeX=Y=Z: 2.56 Å
Density
Contour LevelBy AUTHOR: 2.4 / Movie #1: 2.4
Minimum - Maximum-4.9149437 - 14.35018635
Average (Standard dev.)0.0 (±0.99999976)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 327.68 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.562.562.56
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z327.680327.680327.680
α/β/γ90.00090.00090.000
start NX/NY/NZ-36-30-80
NX/NY/NZ7361161
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-4.91514.3500.000

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Supplemental data

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Sample components

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Entire : apo TFIIH

EntireName: apo TFIIH
Components
  • Sample: apo TFIIH
  • Protein or peptide: General transcription factor IIH

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Supramolecule #1000: apo TFIIH

SupramoleculeName: apo TFIIH / type: sample / ID: 1000 / Number unique components: 1
Molecular weightTheoretical: 450 KDa

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Macromolecule #1: General transcription factor IIH

MacromoleculeName: General transcription factor IIH / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Cell: Hela / Organelle: Nucleus
Molecular weightTheoretical: 450 KDa

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.04 mg/mL
BufferpH: 7.9
Details: 10 mM HEPES, 5% glycerol, 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.05% NP-40
StainingType: NEGATIVE
Details: Grids with adsorbed protein floated on 2% w/v uranyl formate for 1 minute.
GridDetails: 200 mesh Cu grid
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 80000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: 1.2 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 80000
Sample stageSpecimen holder: Room temp single tilt / Specimen holder model: SIDE ENTRY, EUCENTRIC
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 250,000 times magnification.
DetailsData acquired using Leginon
DateApr 17, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 450 / Average electron dose: 20 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: whole micrograph
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: EMAN2, SPARX
Details: Image processing was performed in the Appion processing environment. 3D reconstruction was performed using EMAN2 and SPARX libraries. Final map was filtered to local resolution using the ...Details: Image processing was performed in the Appion processing environment. 3D reconstruction was performed using EMAN2 and SPARX libraries. Final map was filtered to local resolution using the blocres function in Bsoft package.
Number images used: 13023

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