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- EMDB-2136: Negative stain EM composite structure (part 1) of the type IV sec... -

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Entry
Database: EMDB / ID: EMD-2136
TitleNegative stain EM composite structure (part 1) of the type IV secretion system subcomplex VirB4-VirB7-VirB9-VirB10Secretion
Map dataCore complex part of the VirB4-core complex composite reconstruction
Sample
  • Sample: TraB/TraN/TraO/TraF complex encoded by pKM101
  • Protein or peptide: TraB
  • Protein or peptide: TraN
  • Protein or peptide: TraO
  • Protein or peptide: TraFTNF receptor associated factor
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / negative staining / Resolution: 20.0 Å
AuthorsWilliams R / Wallden K / Yan J / Lian PW / Wang L / Thalassinos K / Orlova EV / Waksman G
CitationJournal: Proc Natl Acad Sci U S A / Year: 2012
Title: Structure of the VirB4 ATPase, alone and bound to the core complex of a type IV secretion system.
Authors: Karin Walldén / Robert Williams / Jun Yan / Pei W Lian / Luchun Wang / Konstantinos Thalassinos / Elena V Orlova / Gabriel Waksman /
Abstract: Type IV secretion (T4S) systems mediate the transfer of proteins and DNA across the cell envelope of bacteria. These systems play important roles in bacterial pathogenesis and in horizontal transfer ...Type IV secretion (T4S) systems mediate the transfer of proteins and DNA across the cell envelope of bacteria. These systems play important roles in bacterial pathogenesis and in horizontal transfer of antibiotic resistance. The VirB4 ATPase of the T4S system is essential for both the assembly of the system and substrate transfer. In this article, we present the crystal structure of the C-terminal domain of Thermoanaerobacter pseudethanolicus VirB4. This structure is strikingly similar to that of another T4S ATPase, VirD4, a protein that shares only 12% sequence identity with VirB4. The VirB4 domain purifies as a monomer, but the full-length protein is observed in a monomer-dimer equilibrium, even in the presence of nucleotides and DNAs. We also report the negative stain electron microscopy structure of the core complex of the T4S system of the Escherichia coli pKM101 plasmid, with VirB4 bound. In this structure, VirB4 is also monomeric and bound through its N-terminal domain to the core's VirB9 protein. Remarkably, VirB4 is observed bound to the side of the complex where it is ideally placed to play its known regulatory role in substrate transfer.
History
DepositionJun 12, 2012-
Header (metadata) releaseJul 25, 2012-
Map releaseAug 1, 2012-
UpdateAug 29, 2012-
Current statusAug 29, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.25
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD2136.tif

Downloads & links

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Map

FileDownload / File: emd_2136.map.gz / Format: CCP4 / Size: 58.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCore complex part of the VirB4-core complex composite reconstruction
Voxel sizeX=Y=Z: 1.67 Å
Density
Contour LevelBy EMDB: 0.35 / Movie #1: 0.25
Minimum - Maximum-1.48925638 - 1.98086631
Average (Standard dev.)0.0111697 (±0.09054922)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-125-125-125
Dimensions250250250
Spacing250250250
CellA=B=C: 417.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.671.671.67
M x/y/z250250250
origin x/y/z0.0000.0000.000
length x/y/z417.500417.500417.500
α/β/γ90.00090.00090.000
start NX/NY/NZ-32-32-32
NX/NY/NZ646464
MAP C/R/S123
start NC/NR/NS-125-125-125
NC/NR/NS250250250
D min/max/mean-1.4891.9810.011

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Supplemental data

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Sample components

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Entire : TraB/TraN/TraO/TraF complex encoded by pKM101

EntireName: TraB/TraN/TraO/TraF complex encoded by pKM101
Components
  • Sample: TraB/TraN/TraO/TraF complex encoded by pKM101
  • Protein or peptide: TraB
  • Protein or peptide: TraN
  • Protein or peptide: TraO
  • Protein or peptide: TraFTNF receptor associated factor

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Supramolecule #1000: TraB/TraN/TraO/TraF complex encoded by pKM101

SupramoleculeName: TraB/TraN/TraO/TraF complex encoded by pKM101 / type: sample / ID: 1000
Oligomeric state: 14-mer of core complex (TraN/TraO/TraF) and monomer of TraB
Number unique components: 4
Molecular weightExperimental: 1.15 MDa / Theoretical: 1.15 MDa

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Macromolecule #1: TraB

MacromoleculeName: TraB / type: protein_or_peptide / ID: 1 / Name.synonym: VirB4
Details: The 1.05 MDa core complex (TraN/TraO/TraF) with TraB bound, see EMD-2137
Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Strain: pKM101 plasmid / Location in cell: cell envelope
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pASK IBA3C

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Macromolecule #2: TraN

MacromoleculeName: TraN / type: protein_or_peptide / ID: 2 / Name.synonym: VirB7
Details: The 1.05 MDa core complex (TraN/TraO/TraF) with TraB bound, see EMD-2137
Number of copies: 14 / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Strain: pKM101 plasmid / Location in cell: cell envelope
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pASK IBA3C

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Macromolecule #3: TraO

MacromoleculeName: TraO / type: protein_or_peptide / ID: 3 / Name.synonym: VirB9
Details: The 1.05 MDa core complex (TraN/TraO/TraF) with TraB bound, see EMD-2137
Number of copies: 14 / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Strain: pKM101 plasmid / Location in cell: cell envelope
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pASK IBA3C

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Macromolecule #4: TraF

MacromoleculeName: TraF / type: protein_or_peptide / ID: 4 / Name.synonym: VirB10
Details: The 1.05 MDa core complex (TraN/TraO/TraF) with TraB bound, see EMD-2137
Number of copies: 14 / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Strain: pKM101 plasmid / Location in cell: cell envelope
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pASK IBA3C

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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

StainingType: NEGATIVE / Details: NanoW
GridDetails: Glow-discharged continuous carbon
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: OTHER
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.7 µm / Nominal magnification: 42000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
DateDec 22, 2009
Image recordingCategory: FILM / Film or detector model: GENERIC FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 12 / Bits/pixel: 8
Tilt angle min0
Tilt angle max0

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Image processing

Final two d classificationNumber classes: 330
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMAGIC
Details: This map is part of a composite structure of two maps.
Number images used: 10000

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