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- EMDB-3378: Transcription initiation complex structures elucidate DNA opening... -

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Entry
Database: EMDB / ID: 3378
TitleTranscription initiation complex structures elucidate DNA opening (OC3)
Map dataunsharpened OC3 map
Sampleyeast Pol II transcription initiation complex (open DNA)
  • DNA-directed RNA polymerase IIPolymerase
  • (Transcription Factor ...) x 4
  • TATA-box-binding protein
  • nucleic-acidNucleic acid
KeywordsGene expression / Transcription initiation
Function / homologyTBP domain superfamily / DNA-directed RNA polymerase M, 15kDa subunit, conserved site / Zinc finger, TFIIB-type / Transcription factor TFIIB, cyclin-like domain / Cyclin-like / DNA-directed RNA polymerase RPB5 subunit, eukaryote/virus / RNA polymerase Rpb2, OB-fold / DNA-directed RNA polymerase, subunit 2 / Transcription initiation factor IIA, gamma subunit, C-terminal / Transcription initiation factor IIA, gamma subunit, N-terminal ...TBP domain superfamily / DNA-directed RNA polymerase M, 15kDa subunit, conserved site / Zinc finger, TFIIB-type / Transcription factor TFIIB, cyclin-like domain / Cyclin-like / DNA-directed RNA polymerase RPB5 subunit, eukaryote/virus / RNA polymerase Rpb2, OB-fold / DNA-directed RNA polymerase, subunit 2 / Transcription initiation factor IIA, gamma subunit, C-terminal / Transcription initiation factor IIA, gamma subunit, N-terminal / Transcription initiation factor TFIIE, beta subunit / Transcription factor TFE/TFIIEalpha HTH domain / RNA polymerase, subunit H/Rpb5, conserved site / Nucleic acid-binding, OB-fold / Archaeal RpoH /eukaryotic RPB5 RNA polymerase subunit / DNA-directed RNA polymerase, 14-18kDa subunit, conserved site / RNA polymerases, subunit N, zinc binding site / RNA-binding domain, S1 / Transcription factor TFIIB, conserved site / RNA polymerase subunit RPB10 / TFIIEalpha/SarR/Rpc3 HTH domain / DNA-directed RNA polymerase, subunit RPB6 / RNA polymerase subunit RPABC4/transcription elongation factor Spt4 / TATA-box binding protein, conserved site / TATA-box binding protein, eukaryotic / Zinc finger, RING/FYVE/PHD-type / RNA polymerase subunit, RPB6/omega / RPB5-like RNA polymerase subunit superfamily / RNA polymerase, beta subunit, protrusion / RNA polymerase, N-terminal / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 7 / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / RNA polymerase, beta subunit, conserved site / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 3 / DNA-directed RNA polymerase subunit/transcription factor S / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 5 / DNA-directed RNA polymerase Rpb11, 13-16kDa subunit, conserved site / Transcription initiation factor IIF, alpha subunit / DNA-directed RNA polymerase, RBP11-like dimerisation domain / Transcription factor IIA, helical / Transcription factor IIA, beta-barrel / HRDC-like superfamily / Transcription Factor IIF, Rap30/Rap74, interaction / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / Pol II subunit B9, C-terminal zinc ribbon / RPB6/omega subunit-like superfamily / RNA polymerase Rpb4/RPC9, core / RNA polymerase Rpb4 / RNA polymerases N / 8 kDa subunit / TFIIE alpha subunit / RNA polymerases M/15 Kd subunit / TFIIE beta subunit core domain / Transcription initiation factor IIA, gamma subunit, helical domain / TFIIF, beta subunit HTH domain / Transcription initiation factor IIA, gamma subunit / Transcription factor IIA, alpha/beta subunit / DNA directed RNA polymerase, 7 kDa subunit / RNA polymerase Rpb8 / RNA polymerase Rpb5, N-terminal domain / SHS2 domain found in N terminus of Rpb7p/Rpc25p/MJ0397 / RNA polymerase Rpb6 / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 2 / RNA polymerase beta subunit / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 7 / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerase Rpb5, C-terminal domain / Winged helix-like DNA-binding domain superfamily / RNA polymerase Rpb1, domain 7 superfamily / Winged helix DNA-binding domain superfamily / RNA polymerase, RBP11-like subunit / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase Rpb5, N-terminal domain superfamily / RNA polymerase Rpb7-like, N-terminal domain superfamily / Cyclin-like superfamily / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase RBP11 / RNA polymerase Rpb1, funnel domain superfamily / Rpb4/RPC9 superfamily / DNA-directed RNA polymerase subunit Rpb5-like / Transcription factor S-II (TFIIS) / DNA-directed RNA polymerases I, II, and III subunit RPABC4 / Transcription factor E
Function and homology information
SourceSaccharomyces cerevisiae (baker's yeast) / unidentified (others)
Methodsingle particle reconstruction / cryo EM / 4.35 Å resolution
AuthorsPlaschka C / Hantsche M / Dienemann C / Burzinski C / Plitzko J / Cramer P
CitationJournal: Nature / Year: 2016
Title: Transcription initiation complex structures elucidate DNA opening.
Authors: C Plaschka / M Hantsche / C Dienemann / C Burzinski / J Plitzko / P Cramer
Validation ReportPDB-ID: 5fyw

SummaryFull reportAbout validation report
DateDeposition: Mar 9, 2016 / Header (metadata) release: Apr 13, 2016 / Map release: May 18, 2016 / Last update: Jun 15, 2016

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0325
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0325
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-5fyw
  • Surface level: 0.0325
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_3378.map.gz (map file in CCP4 format, 105470 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
300 pix
1.35 Å/pix.
= 405. Å
300 pix
1.35 Å/pix.
= 405. Å
300 pix
1.35 Å/pix.
= 405. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.35 Å
Density
Contour Level:0.0325 (by author), 0.0325 (movie #1):
Minimum - Maximum-0.03927520 - 0.09277353
Average (Standard dev.)-0.00005954 (0.00432831)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions300300300
Origin000
Limit299299299
Spacing300300300
CellA=B=C: 405.0 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z405.000405.000405.000
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0390.093-0.000

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Supplemental data

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Sample components

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Entire yeast Pol II transcription initiation complex (open DNA)

EntireName: yeast Pol II transcription initiation complex (open DNA)
Number of components: 7 / Oligomeric State: Monomer
MassTheoretical: 890 kDa

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Component #1: protein, DNA-directed RNA polymerase II

ProteinName: DNA-directed RNA polymerase IIPolymerase / a.k.a: RNA polymerase II / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 513 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast) / Strain: BJ5464 Rpb3 His-Bio

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Component #2: protein, Transcription Factor IIA

ProteinName: Transcription Factor IIA / a.k.a: TFIIATranscription factor II A / Number of Copies: 1 / Recombinant expression: Yes
MassTheoretical: 45 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pOPINE / Strain: BL21(DE3)RIL

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Component #3: protein, Transcription Factor IIB

ProteinName: Transcription Factor IIBTranscription factor II B / a.k.a: TFIIBTranscription factor II B / Recombinant expression: Yes / Number of Copies: 1
MassTheoretical: 38 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pET21b / Strain: BL21(DE3)RIL

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Component #4: protein, TATA-box-binding protein

ProteinName: TATA-box-binding protein / a.k.a: TBP, SPT15 / Number of Copies: 1 / Recombinant expression: Yes
MassTheoretical: 27 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pOPINE / Strain: BL21(DE3)RIL

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Component #5: protein, Transcription Factor IIE

ProteinName: Transcription Factor IIE / a.k.a: TFIIETranscription factor II E / Number of Copies: 1 / Recombinant expression: Yes
MassTheoretical: 91 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pET21 / Strain: BL21(DE3)RIL

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Component #6: protein, Transcription Factor IIF

ProteinName: Transcription Factor IIF / a.k.a: TFIIFTranscription factor II F / Recombinant expression: Yes / Number of Copies: 1
MassTheoretical: 129 kDa
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pETDuet-1 / Strain: BL21(DE3)RIL

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Component #7: nucleic-acid, synthetic promoter DNA construct (open)

Nucleic-acidName: synthetic promoter DNA construct (open) / Class: DNA / Details: Contains a 15 nucleotide mismatch bubble / Structure: DOUBLE HELIX / Synthetic: Yes
MassTheoretical: 44 kDa
SourceSpecies: unidentified (others)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.3 mg/ml
Buffer solution: 25 mM HEPES-KOH pH 7.5, 150 mM potassium acetate, 2 mM MgCl2, 5 mM DTT
pH: 7.5
Support filmR3.5/1 holey carbon grids (Quantifoil)
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %
Method: Quantifoil R 3.5/1 holey carbon grids were glow-discharged before deposition of 4.5 microliters of sample. Grids were then blotted for 8.5 s and plunge-frozen in liquid ethane.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Apr 26, 2016
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 33 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 37000 X (nominal), 37037 X (calibrated) / Cs: 2.3 mm / Imaging mode: BRIGHT FIELD / Defocus: 700 - 4200 nm / Energy filter: GIF Quantum / Energy window: 0-20 eV
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 1756

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 11231
3D reconstructionAlgorithm: RELION 1.3 / Software: RELION / CTF correction: Each particle / Resolution: 4.35 Å / Resolution method: FSC 0.143, gold-standard

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