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- EMDB-5025: Cryo-EM structure of Pyrococcus furiosus pre-initiation complex -

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Basic information

Entry
Database: EMDB / ID: EMD-5025
TitleCryo-EM structure of Pyrococcus furiosus pre-initiation complex
Map dataArchaea RNAP TBP TFB DNA initiation complex
Sample
  • Sample: Archaea RNAP TBP TFB DNA initiation complex
  • Protein or peptide: RNAP PIC
Keywordstranscription / initiation / elongation / RNAP / Archaea / cryo-electron microscopy / negative staining / molecular fitting
Biological speciesunidentified (others)
Methodsingle particle reconstruction / negative staining / Resolution: 25.0 Å
AuthorsCarlo SD / Lin S-C / Taatjes D / Hoenger A
CitationJournal: Transcription / Year: 2010
Title: Molecular basis of transcription initiation in Archaea.
Authors: Sacha De Carlo / Shih-Chieh Lin / Dylan J Taatjes / Andreas Hoenger /
Abstract: Compared with eukaryotes, the archaeal transcription initiation machinery-commonly known as the Pre-Initiation Complex-is relatively simple. The archaeal PIC consists of the TFIIB ortholog TFB, TBP, ...Compared with eukaryotes, the archaeal transcription initiation machinery-commonly known as the Pre-Initiation Complex-is relatively simple. The archaeal PIC consists of the TFIIB ortholog TFB, TBP, and an 11-subunit RNA polymerase (RNAP). The relatively small size of the entire archaeal PIC makes it amenable to structural analysis. Using purified RNAP, TFB, and TBP from the thermophile Pyrococcus furiosus, we assembled the biochemically active PIC at 65ºC. The intact archaeal PIC was isolated by implementing a cross-linking technique followed by size-exclusion chromatography, and the structure of this 440 kDa assembly was determined using electron microscopy and single-particle reconstruction techniques. Combining difference maps with crystal structure docking of various sub-domains, TBP and TFB were localized within the macromolecular PIC. TBP/TFB assemble near the large RpoB subunit and the RpoD/L "foot" domain behind the RNAP central cleft. This location mimics that of yeast TBP and TFIIB in complex with yeast RNAP II. Collectively, these results define the structural organization of the archaeal transcription machinery and suggest a conserved core PIC architecture.
History
DepositionJul 30, 2008-
Header (metadata) releaseDec 17, 2008-
Map releaseMar 29, 2010-
UpdateJul 17, 2013-
Current statusJul 17, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5025_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5025.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationArchaea RNAP TBP TFB DNA initiation complex
Voxel sizeX=Y=Z: 2.25 Å
Density
Contour LevelBy AUTHOR: 0.017 / Movie #1: 0.015
Minimum - Maximum-0.02186078 - 0.05420369
Average (Standard dev.)0.00017935 (±0.00381124)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 450.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.252.252.25
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z450.000450.000450.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-127-127-127
NX/NY/NZ255255255
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0220.0540.000

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Supplemental data

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Sample components

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Entire : Archaea RNAP TBP TFB DNA initiation complex

EntireName: Archaea RNAP TBP TFB DNA initiation complex
Components
  • Sample: Archaea RNAP TBP TFB DNA initiation complex
  • Protein or peptide: RNAP PIC

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Supramolecule #1000: Archaea RNAP TBP TFB DNA initiation complex

SupramoleculeName: Archaea RNAP TBP TFB DNA initiation complex / type: sample / ID: 1000 / Oligomeric state: Quaternary complex / Number unique components: 4
Molecular weightExperimental: 440 KDa / Method: Mass spectrometry, gel-filtration, SDS-PAGE

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Macromolecule #1: RNAP PIC

MacromoleculeName: RNAP PIC / type: protein_or_peptide / ID: 1 / Name.synonym: Pre-Initiation Complex / Number of copies: 1 / Oligomeric state: Complex / Recombinant expression: No / Database: NCBI
Source (natural)Organism: unidentified (others) / Cell: Pyrococcus
Molecular weightExperimental: 440 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

Concentration0.05 mg/mL
BufferDetails: Hepes 0.1 M, pH 8.0, NaCl 50 mM, EDTA 0.1mM, DTT 2 mM
StainingType: NEGATIVE / Details: 2% w/v uranyl acetate for 30 seconds.
GridDetails: Continuous carbon grids
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 49785 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal magnification: 50000
Sample stageSpecimen holder: single-tilt / Specimen holder model: OTHER
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 6.7 µm / Number real images: 36 / Average electron dose: 18 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER, EMAN / Number images used: 5582
Detailscross-linked in the presence of 0.05% glutaraldehyde

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Atomic model buiding 1

Initial modelPDB ID:

2pmz

SoftwareName: CHIMERA
DetailsProtocol: Rigid Body
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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