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- EMDB-2308: Structural visualization of key steps in human transcription init... -

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Basic information

Entry
Database: EMDB / ID: EMD-2308
TitleStructural visualization of key steps in human transcription initiation
Map dataTBP-TFIIA-TFIIB-DNA-Pol II-TFIIF-TFIIE-TFIIH (close)
Sample
  • Sample: TBP-TFIIA-TFIIB-DNA-Pol II-TFIIF-TFIIE-TFIIH (close)
  • Protein or peptide: TATA-box-binding protein
  • Protein or peptide: General transcription factor IIA
  • Protein or peptide: General transcription factor IIB
  • Protein or peptide: General transcription factor IIF
  • Protein or peptide: General transcription factor IIE
  • Protein or peptide: General transcription factor IIH
  • Protein or peptide: DNA-directed RNA polymerase II
  • DNA: Core promoter DNA
Keywordshuman transcription initiation
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 14.0 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: 1.69
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 1.69
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2308.png

Downloads & links

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Map

FileDownload / File: emd_2308.map.gz / Format: CCP4 / Size: 15.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTBP-TFIIA-TFIIB-DNA-Pol II-TFIIF-TFIIE-TFIIH (close)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.56 Å/pix.
x 160 pix.
= 409.6 Å		2.56 Å/pix.
x 160 pix.
= 409.6 Å		2.56 Å/pix.
x 160 pix.
= 409.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.56 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.69 / Movie #1: 1.69
Minimum - Maximum-12.55161858 - 13.810432430000001
Average (Standard dev.)0.00442404 (±0.78436887)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 409.59998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.562.562.56
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z409.600409.600409.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-36-30-80
NX/NY/NZ7361161
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-12.55213.8100.004

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Supplemental data

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Sample components

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Entire : TBP-TFIIA-TFIIB-DNA-Pol II-TFIIF-TFIIE-TFIIH (close)

EntireName: TBP-TFIIA-TFIIB-DNA-Pol II-TFIIF-TFIIE-TFIIH (close)
Components
  • Sample: TBP-TFIIA-TFIIB-DNA-Pol II-TFIIF-TFIIE-TFIIH (close)
  • Protein or peptide: TATA-box-binding protein
  • Protein or peptide: General transcription factor IIA
  • Protein or peptide: General transcription factor IIB
  • Protein or peptide: General transcription factor IIF
  • Protein or peptide: General transcription factor IIE
  • Protein or peptide: General transcription factor IIH
  • Protein or peptide: DNA-directed RNA polymerase II
  • DNA: Core promoter DNA

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Supramolecule #1000: TBP-TFIIA-TFIIB-DNA-Pol II-TFIIF-TFIIE-TFIIH (close)

SupramoleculeName: TBP-TFIIA-TFIIB-DNA-Pol II-TFIIF-TFIIE-TFIIH (close) / type: sample / ID: 1000 / Number unique components: 8
Molecular weightTheoretical: 1.35 MDa

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Macromolecule #1: TATA-box-binding protein

MacromoleculeName: TATA-box-binding protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 38 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #2: General transcription factor IIA

MacromoleculeName: General transcription factor IIA / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 54 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #3: General transcription factor IIB

MacromoleculeName: General transcription factor IIB / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 35 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #4: General transcription factor IIF

MacromoleculeName: General transcription factor IIF / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 87 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #5: General transcription factor IIE

MacromoleculeName: General transcription factor IIE / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 82 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #6: General transcription factor IIH

MacromoleculeName: General transcription factor IIH / type: protein_or_peptide / ID: 6 / 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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Macromolecule #7: DNA-directed RNA polymerase II

MacromoleculeName: DNA-directed RNA polymerase II / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Cell: Hela / Organelle: Nucleus
Molecular weightTheoretical: 550 KDa

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Macromolecule #8: Core promoter DNA

MacromoleculeName: Core promoter DNA / type: dna / ID: 8 / Classification: DNA / Structure: DOUBLE HELIX / Synthetic?: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: human
Molecular weightTheoretical: 55 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

Concentration0.06 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
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 250,000 times magnification.
DetailsData acquired using Leginon
DateApr 15, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 600 / Average electron dose: 20 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 80000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / 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
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: 14.0 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: 64712

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