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- EMDB-2637: Negative stain electron microscopy of Bacillus subtilis RNA polym... -

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Basic information

Entry
Database: EMDB / ID: EMD-2637
TitleNegative stain electron microscopy of Bacillus subtilis RNA polymerase with YkzG-GFP fusion
Map dataReconstruction of B. subtilis RNAP with YkzG-GFP fusion
Sample
  • Sample: RNA polymerase YkzG-GFP
  • Protein or peptide: RNA polymerase
KeywordsRNA polymerase / small subunit / transcription / Bacillus subtilis
Biological speciesBacillus subtilis (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 24.0 Å
AuthorsKeller A / Yang X / Korelusova J / Delumeau O / Krasny L / Lewis PJ
CitationJournal: J Bacteriol / Year: 2014
Title: ε, a new subunit of RNA polymerase found in gram-positive bacteria.
Authors: Andrew N Keller / Xiao Yang / Jana Wiedermannová / Olivier Delumeau / Libor Krásný / Peter J Lewis /
Abstract: RNA polymerase in bacteria is a multisubunit protein complex that is essential for gene expression. We have identified a new subunit of RNA polymerase present in the high-A+T Firmicutes phylum of ...RNA polymerase in bacteria is a multisubunit protein complex that is essential for gene expression. We have identified a new subunit of RNA polymerase present in the high-A+T Firmicutes phylum of Gram-positive bacteria and have named it ε. Previously ε had been identified as a small protein (ω1) that copurified with RNA polymerase. We have solved the structure of ε by X-ray crystallography and show that it is not an ω subunit. Rather, ε bears remarkable similarity to the Gp2 family of phage proteins involved in the inhibition of host cell transcription following infection. Deletion of ε shows no phenotype and has no effect on the transcriptional profile of the cell. Determination of the location of ε within the assembly of RNA polymerase core by single-particle analysis suggests that it binds toward the downstream side of the DNA binding cleft. Due to the structural similarity of ε with Gp2 and the fact they bind similar regions of RNA polymerase, we hypothesize that ε may serve a role in protection from phage infection.
History
DepositionApr 29, 2014-
Header (metadata) releaseMay 28, 2014-
Map releaseAug 20, 2014-
UpdateOct 1, 2014-
Current statusOct 1, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.2
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2637.png

emd-2637.png

Downloads & links

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Map

FileDownload / File: emd_2637.map.gz / Format: CCP4 / Size: 1.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of B. subtilis RNAP with YkzG-GFP fusion
Voxel sizeX=Y=Z: 3.6 Å
Density
Contour LevelBy AUTHOR: 1.2 / Movie #1: 1.2
Minimum - Maximum-4.09110546 - 8.46971989
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-10-10-10
Dimensions727272
Spacing727272
CellA=B=C: 259.19998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.63.63.6
M x/y/z727272
origin x/y/z0.0000.0000.000
length x/y/z259.200259.200259.200
α/β/γ90.00090.00090.000
start NX/NY/NZ0-51-100
NX/NY/NZ82103201
MAP C/R/S123
start NC/NR/NS-10-10-10
NC/NR/NS727272
D min/max/mean-4.0918.470-0.000

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Supplemental data

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Sample components

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Entire : RNA polymerase YkzG-GFP

EntireName: RNA polymerase YkzG-GFP
Components
  • Sample: RNA polymerase YkzG-GFP
  • Protein or peptide: RNA polymerase

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Supramolecule #1000: RNA polymerase YkzG-GFP

SupramoleculeName: RNA polymerase YkzG-GFP / type: sample / ID: 1000 / Number unique components: 1
Molecular weightExperimental: 430 KDa / Theoretical: 430 KDa

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Macromolecule #1: RNA polymerase

MacromoleculeName: RNA polymerase / type: protein_or_peptide / ID: 1 / Name.synonym: RNAP / Details: RNAP with YkzG-GFP fusion / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Bacillus subtilis (bacteria) / Strain: BS509
Molecular weightExperimental: 430 KDa / Theoretical: 430 KDa
Recombinant expressionOrganism: Bacillus subtilis (bacteria)

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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

Concentration1 mg/mL
BufferpH: 7.8
Details: 20 mM Tris-HCl , 150 mM NaCl, 10 mM MgCl2, 5% (v/v) glycerol and 1 mM DTT
StainingType: NEGATIVE
Details: The specimens were then stained with a 1 % (w/v) uranyl formate aqueous solution.
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F30
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: JEOL
DateDec 1, 2009
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 260
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 24.0 Å / Resolution method: OTHER / Software - Name: EMAN2.8 / Number images used: 10104

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