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- EMDB-1567: Sigma 54 RNA polymerase holoenzyme -

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Basic information

Entry
Database: EMDB / ID: EMD-1567
TitleSigma 54 RNA polymerase holoenzyme
Map dataSigma 54 RNA polymerase holoenzyme
Sample
  • Sample: Complex of E.coli RNA polymerase and Sigma 54
  • Protein or peptide: RNA polymerase
  • Protein or peptide: Sigma 54
KeywordsRNA polymerase / RNAP / Sigma 54 / transcription initiation / sigma factor
Biological speciesEscherichia coli (E. coli) / Klebsiella pneumoniae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 21.0 Å
AuthorsBose D / Pape T / Burrows PC / Rappas M / Wigneshweraraj SR / Buck M / Zhang X
CitationJournal: Mol Cell / Year: 2008
Title: Organization of an activator-bound RNA polymerase holoenzyme.
Authors: Daniel Bose / Tillmann Pape / Patricia C Burrows / Mathieu Rappas / Siva R Wigneshweraraj / Martin Buck / Xiaodong Zhang /
Abstract: Transcription initiation involves the conversion from closed promoter complexes, comprising RNA polymerase (RNAP) and double-stranded promoter DNA, to open complexes, in which the enzyme is able to ...Transcription initiation involves the conversion from closed promoter complexes, comprising RNA polymerase (RNAP) and double-stranded promoter DNA, to open complexes, in which the enzyme is able to access the DNA template in a single-stranded form. The complex between bacterial RNAP and its major variant sigma factor sigma(54) remains as a closed complex until ATP hydrolysis-dependent remodeling by activator proteins occurs. This remodeling facilitates DNA melting and allows the transition to the open complex. Here we present cryoelectron microscopy reconstructions of bacterial RNAP in complex with sigma(54) alone, and of RNAP-sigma(54) with an AAA+ activator. Together with photo-crosslinking data that establish the location of promoter DNA within the complexes, we explain why the RNAP-sigma(54) closed complex is unable to access the DNA template and propose how the structural changes induced by activator binding can initiate conformational changes that ultimately result in formation of the open complex.
History
DepositionOct 1, 2008-
Header (metadata) releaseOct 3, 2008-
Map releaseApr 1, 2009-
UpdateOct 24, 2012-
Current statusOct 24, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0181
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0181
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1567.gif

Downloads & links

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Map

FileDownload / File: emd_1567.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSigma 54 RNA polymerase holoenzyme
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesY (Sec.)X (Row.)Z (Col.)
2.6 Å/pix.
x 128 pix.
= 332.8 Å		2.6 Å/pix.
x 128 pix.
= 332.8 Å		2.6 Å/pix.
x 128 pix.
= 332.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.6 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0181 / Movie #1: 0.0181
Minimum - Maximum-0.211228 - 0.298759
Average (Standard dev.)-0.000000000000223 (±0.0121114)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZXY
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 332.8 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.62.62.6
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z332.800332.800332.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S312
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-0.2110.299-0.000

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Supplemental data

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Sample components

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Entire : Complex of E.coli RNA polymerase and Sigma 54

EntireName: Complex of E.coli RNA polymerase and Sigma 54
Components
  • Sample: Complex of E.coli RNA polymerase and Sigma 54
  • Protein or peptide: RNA polymerase
  • Protein or peptide: Sigma 54

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Supramolecule #1000: Complex of E.coli RNA polymerase and Sigma 54

SupramoleculeName: Complex of E.coli RNA polymerase and Sigma 54 / type: sample / ID: 1000 / Details: All components are present on SDS-PAGE. / Oligomeric state: monomer of RNAP sigma 54 holoenzyme / Number unique components: 2
Molecular weightTheoretical: 450 KDa

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Macromolecule #1: RNA polymerase

MacromoleculeName: RNA polymerase / type: protein_or_peptide / ID: 1 / Name.synonym: RNAP / Number of copies: 1 / Oligomeric state: pentamer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 390 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pvs10

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Macromolecule #2: Sigma 54

MacromoleculeName: Sigma 54 / type: protein_or_peptide / ID: 2 / Name.synonym: s45 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Molecular weightTheoretical: 54 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: PET28b

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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

BufferpH: 8 / Details: 10mM Tris-HCl pH8.0, 150mM NaCl, 10mM MgCl2,
GridDetails: Copper 400 mesh
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 83 K / Instrument: OTHER / Details: Vitrification instrument: Vitrobot

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG/UT
TemperatureAverage: 91 K
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 6.35 µm / Number real images: 50 / Average electron dose: 10 e/Å2 / Od range: 4.8 / Bits/pixel: 16
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 48700 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.1 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder: eucentric / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

CTF correctionDetails: particles
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 21.0 Å / Resolution method: OTHER / Software - Name: Imagic V / Number images used: 10320
Final two d classificationNumber classes: 516

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Atomic model buiding 1

Initial modelPDB ID:

1iw7

SoftwareName: Situs
DetailsProtocol: Rigid body. Structure positioned by hand and fitting refined in Situs
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 0.7

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