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- EMDB-5505: Hexameric structure of the conjugative VirB4 ATPase TrwK: new ins... -

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Basic information

Entry
Database: EMDB / ID: EMD-5505
TitleHexameric structure of the conjugative VirB4 ATPase TrwK: new insights into a functional and phylogenetic relationship with DNA translocases
Map dataReconstruction of hexameric TrwK
Sample
  • Sample: Hexameric TrwK
  • Protein or peptide: VirB4 component of type IV transporter system
Keywordsbacterial secretion systems / conjugation
Function / homologyCagE, TrbE, VirB component of type IV transporter system / ATP binding
Function and homology information
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / negative staining / Resolution: 20.0 Å
AuthorsPena A / Matilla I / Martin-Benito J / Valpuesta JM / Carrascosa JL / De la Cruz F / Cabezon E / Arechaga I
CitationJournal: J Biol Chem / Year: 2012
Title: The hexameric structure of a conjugative VirB4 protein ATPase provides new insights for a functional and phylogenetic relationship with DNA translocases.
Authors: Alejandro Peña / Inmaculada Matilla / Jaime Martín-Benito / José M Valpuesta / José L Carrascosa / Fernando de la Cruz / Elena Cabezón / Ignacio Arechaga /
Abstract: VirB4 proteins are ATPases essential for pilus biogenesis and protein transport in type IV secretion systems. These proteins contain a motor domain that shares structural similarities with the motor ...VirB4 proteins are ATPases essential for pilus biogenesis and protein transport in type IV secretion systems. These proteins contain a motor domain that shares structural similarities with the motor domains of DNA translocases, such as the VirD4/TrwB conjugative coupling proteins and the chromosome segregation pump FtsK. Here, we report the three-dimensional structure of full-length TrwK, the VirB4 homologue in the conjugative plasmid R388, determined by single-particle electron microscopy. The structure consists of a hexameric double ring with a barrel-shaped structure. The C-terminal half of VirB4 proteins shares a striking structural similarity with the DNA translocase TrwB. Docking the atomic coordinates of the crystal structures of TrwB and FtsK into the EM map revealed a better fit for FtsK. Interestingly, we have found that like TrwB, TrwK is able to bind DNA with a higher affinity for G4 quadruplex structures than for single-stranded DNA. Furthermore, TrwK exerts a dominant negative effect on the ATPase activity of TrwB, which reflects an interaction between the two proteins. Our studies provide new insights into the structure-function relationship and the evolution of these DNA and protein translocases.
History
DepositionSep 28, 2012-
Header (metadata) releaseOct 10, 2012-
Map releaseOct 24, 2012-
UpdateNov 28, 2012-
Current statusNov 28, 2012Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5505.tif

Downloads & links

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Map

FileDownload / File: emd_5505.map.gz / Format: CCP4 / Size: 602.5 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of hexameric TrwK
Voxel sizeX=Y=Z: 4.66 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.07715909 - 0.11468405
Average (Standard dev.)0.00622329 (±0.02549224)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions545454
Spacing545454
CellA=B=C: 251.63998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.664.664.66
M x/y/z545454
origin x/y/z0.0000.0000.000
length x/y/z251.640251.640251.640
α/β/γ90.00090.00090.000
start NX/NY/NZ-5029166
NX/NY/NZ106122134
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS545454
D min/max/mean-0.0770.1150.006

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Supplemental data

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Sample components

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Entire : Hexameric TrwK

EntireName: Hexameric TrwK
Components
  • Sample: Hexameric TrwK
  • Protein or peptide: VirB4 component of type IV transporter system

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Supramolecule #1000: Hexameric TrwK

SupramoleculeName: Hexameric TrwK / type: sample / ID: 1000 / Oligomeric state: hexameric / Number unique components: 1
Molecular weightTheoretical: 540 KDa

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Macromolecule #1: VirB4 component of type IV transporter system

MacromoleculeName: VirB4 component of type IV transporter system / type: protein_or_peptide / ID: 1 / Name.synonym: VirB4 / Oligomeric state: Hexamer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 540 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET
SequenceGO: ATP binding
InterPro: CagE, TrbE, VirB component of type IV transporter system

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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

Concentration0.1 mg/mL
BufferpH: 6.45
Details: 50 mM PIPES-NaOH, 75 mM potassium acetate, 5% (w/v) glycerol, 10 mM magnesium acetate, 0.1 mM EDTA, and 0.5 mM phenylmethylsulfonyl fluoride.
StainingType: NEGATIVE
Details: Samples were negatively stained with 2% (w/v) uranyl acetate
GridDetails: freshly glow-discharged carbon-coated grids
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeJEOL 1200EXII
Electron beamAcceleration voltage: 100 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 5.6 mm / Nominal defocus max: 1.0 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 60000
Sample stageSpecimen holder model: JEOL
DateDec 11, 2011
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Average electron dose: 20 e/Å2

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Image processing

CTF correctionDetails: Yes
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: XMIPP / Number images used: 1179

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