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- EMDB-5024: Cryo-EM structure of Pyrococcus furiosus in apo-state -

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Basic information

Entry
Database: EMDB / ID: EMD-5024
TitleCryo-EM structure of Pyrococcus furiosus in apo-state
Map dataArchaea RNAP in its (transcription) elongation state.
Sample
  • Sample: Pyrococcus furiosus RNAP
  • Protein or peptide: RNAP apo-state
Keywordstranscription / initiation / elongation / RNAP / Archaea / cryo-electron microscopy / negative staining / molecular fitting
Biological speciesunidentified (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 18.0 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: 0.012
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.012
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5024_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5024.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationArchaea RNAP in its (transcription) elongation state.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.25 Å/pix.
x 200 pix.
= 450. Å		2.25 Å/pix.
x 200 pix.
= 450. Å		2.25 Å/pix.
x 200 pix.
= 450. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.25 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.012
Minimum - Maximum-0.00729787 - 0.03152668
Average (Standard dev.)0.00024728 (±0.00241341)
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-0.0070.0320.000

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Supplemental data

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Sample components

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Entire : Pyrococcus furiosus RNAP

EntireName: Pyrococcus furiosus RNAP
Components
  • Sample: Pyrococcus furiosus RNAP
  • Protein or peptide: RNAP apo-state

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Supramolecule #1000: Pyrococcus furiosus RNAP

SupramoleculeName: Pyrococcus furiosus RNAP / type: sample / ID: 1000 / Oligomeric state: RNAP and DNA / Number unique components: 2
Molecular weightExperimental: 380 KDa / Method: Mass spectrometry

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Macromolecule #1: RNAP apo-state

MacromoleculeName: RNAP apo-state / type: protein_or_peptide / ID: 1 / Name.synonym: RNAP elongation complex / Number of copies: 1 / Oligomeric state: multi-subunit complex / Recombinant expression: No / Database: NCBI
Source (natural)Organism: unidentified (others) / Cell: Pyrococcus
Molecular weightExperimental: 380 KDa

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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

Concentration0.5 mg/mL
BufferDetails: Hepes 0.1 M, pH 8.0, NaCl 50 mM, EDTA 0.1mM, DTT 2 mM
GridDetails: C-Flat
VitrificationCryogen name: ETHANE / Chamber humidity: 65 % / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
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: 32 / Average electron dose: 15 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 49785 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal magnification: 50000
Sample stageSpecimen holder: 626 / Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: phase-flipped
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 18.0 Å / Resolution method: OTHER / Software - Name: SPIDER, EMAN / Number images used: 4611

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Atomic model buiding 1

Initial modelPDB ID:

2pmz

SoftwareName: NORMA
DetailsProtocol: Rigid Body. Manual fitting with CHIMERA first.
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT

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