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- EMDB-1109: Structural insights into the activity of enhancer-binding proteins. -

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Basic information

Entry
Database: EMDB / ID: EMD-1109
TitleStructural insights into the activity of enhancer-binding proteins.
Map datathis is a volume map of PspF in complex with sigma54
Sample
  • Sample: PspF AAA domain
  • Protein or peptide: PspF AAA domain
  • Protein or peptide: sigma54
Function / homologyRNA polymerase sigma factor 54 / AAA+ ATPase domain
Function and homology information
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 20.0 Å
AuthorsRappas M / Schumacher J / Beuron F / Niwa H / Bordes P / Wigneshweraraj S / Keetch CA / Robinson CV / Buck M / Zhang X
CitationJournal: Science / Year: 2005
Title: Structural insights into the activity of enhancer-binding proteins.
Authors: Mathieu Rappas / Jorg Schumacher / Fabienne Beuron / Hajime Niwa / Patricia Bordes / Sivaramesh Wigneshweraraj / Catherine A Keetch / Carol V Robinson / Martin Buck / Xiaodong Zhang /
Abstract: Activators of bacterial sigma54-RNA polymerase holoenzyme are mechanochemical proteins that use adenosine triphosphate (ATP) hydrolysis to activate transcription. We have determined by cryogenic ...Activators of bacterial sigma54-RNA polymerase holoenzyme are mechanochemical proteins that use adenosine triphosphate (ATP) hydrolysis to activate transcription. We have determined by cryogenic electron microscopy (cryo-EM) a 20 angstrom resolution structure of an activator, phage shock protein F [PspF(1-275)], which is bound to an ATP transition state analog in complex with its basal factor, sigma54. By fitting the crystal structure of PspF(1-275) at 1.75 angstroms into the EM map, we identified two loops involved in binding sigma54. Comparing enhancer-binding structures in different nucleotide states and mutational analysis led us to propose nucleotide-dependent conformational changes that free the loops for association with sigma54.
History
DepositionFeb 8, 2005-
Header (metadata) releaseFeb 8, 2005-
Map releaseApr 26, 2005-
UpdateMay 26, 2011-
Current statusMay 26, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.26
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.26
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1109.gif

Downloads & links

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Map

FileDownload / File: emd_1109.map.gz / Format: CCP4 / Size: 12.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationthis is a volume map of PspF in complex with sigma54
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2 Å/pix.
x 150 pix.
= 300. Å		2 Å/pix.
x 150 pix.
= 300. Å		2 Å/pix.
x 150 pix.
= 300. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 0.157 / Movie #1: 0.26
Minimum - Maximum-1.00865 - 3.63096
Average (Standard dev.)0.0112357 (±0.146356)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-75-75-75
Dimensions150150150
Spacing150150150
CellA=B=C: 300 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z222
M x/y/z150150150
origin x/y/z0.0000.0000.000
length x/y/z300.000300.000300.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-80-80-80
NX/NY/NZ160160160
MAP C/R/S123
start NC/NR/NS-75-75-75
NC/NR/NS150150150
D min/max/mean-1.0093.6310.011

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Supplemental data

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Sample components

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Entire : PspF AAA domain

EntireName: PspF AAA domain
Components
  • Sample: PspF AAA domain
  • Protein or peptide: PspF AAA domain
  • Protein or peptide: sigma54

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Supramolecule #1000: PspF AAA domain

SupramoleculeName: PspF AAA domain / type: sample / ID: 1000
Oligomeric state: one hexamer of PspF binds to a monomer of sigma54
Number unique components: 2
Molecular weightExperimental: 240 KDa / Theoretical: 240 KDa / Method: mass spec

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Macromolecule #1: PspF AAA domain

MacromoleculeName: PspF AAA domain / type: protein_or_peptide / ID: 1 / Name.synonym: pspf AAA domain / Number of copies: 6 / Oligomeric state: hexamer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / synonym: E Coli
Molecular weightExperimental: 31 KDa / Theoretical: 31 KDa
Recombinant expressionOrganism: Escherichia coli B834 / Recombinant plasmid: pET28 bplus
SequenceInterPro: AAA+ ATPase domain

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Macromolecule #2: sigma54

MacromoleculeName: sigma54 / type: protein_or_peptide / ID: 2 / Name.synonym: sigma54 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / synonym: E Coli
Molecular weightExperimental: 54 KDa / Theoretical: 54 KDa
Recombinant expressionOrganism: B834 / Recombinant plasmid: pET28 bplus
SequenceInterPro: RNA polymerase sigma factor 54

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.05 mg/mL
BufferpH: 8
Details: 10 mM Tris HCl, pH8, 50 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 5% glycerol
StainingType: NEGATIVE
Details: Grids with native sample quench frozen in liquid ethane cooled at -186 C
GridDetails: Holey carbon grids from Agar
VitrificationCryogen name: ETHANE / Chamber temperature: 87.15 K / Timed resolved state: vitrified 30msec after / Method: blot for 2 seconds

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG/UT
TemperatureAverage: 103 K
Detailsweak beam illumination
DateApr 4, 2002
Image recordingCategory: CCD / Film or detector model: KODAK SO-163 FILM / Digitization - Sampling interval: 2 µm / Number real images: 9 / Average electron dose: 20 e/Å2 / Bits/pixel: 14
Electron beamAcceleration voltage: 160 kV / Electron source: LAB6
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: OTHER / Cs: 1.2 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.4 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 3 SIGMA CUT-OFF / Software - Name: IMAGIC-5
Details: Final maps were calculate from 123 class averages from one dataset
Number images used: 3895
Final two d classificationNumber classes: 123

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Atomic model buiding 1

DetailsProtocol: Cross correlation coefficient between projections of fitted model and those from the EM reconstruction. The monomer was manually fitted using the program "O"; the model was p6 symmetrised and individually subunits were readjusted manually using "O"
RefinementProtocol: RIGID BODY FIT

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