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- EMDB-3585: Structure of the VirD4 bound to a Type IV secretion system -

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Basic information

Entry
Database: EMDB / ID: EMD-3585
TitleStructure of the VirD4 bound to a Type IV secretion systemSecretion
Map dataNegative stain electron microscopy of the T4SS3-10 D4 complex enableslocalization of the VirD4 coupling protein within the complex.
Sample
  • Complex: Type IV secretion system, inner membrane complexSecretion
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / negative staining / Resolution: 28.0 Å
AuthorsRedzej A / Ukleja M
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust098302 United Kingdom
CitationJournal: EMBO J / Year: 2017
Title: Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery.
Authors: Adam Redzej / Marta Ukleja / Sarah Connery / Martina Trokter / Catarina Felisberto-Rodrigues / Adam Cryar / Konstantinos Thalassinos / Richard D Hayward / Elena V Orlova / Gabriel Waksman /
Abstract: Type IV secretion (T4S) systems are versatile bacterial secretion systems mediating transport of protein and/or DNA T4S systems are generally composed of 11 VirB proteins and 1 VirD protein (VirD4). ...Type IV secretion (T4S) systems are versatile bacterial secretion systems mediating transport of protein and/or DNA T4S systems are generally composed of 11 VirB proteins and 1 VirD protein (VirD4). The VirB1-11 proteins assemble to form a secretion machinery and a pilus while the VirD4 protein is responsible for substrate recruitment. The structure of VirD4 in isolation is known; however, its structure bound to the VirB1-11 apparatus has not been determined. Here, we purify a T4S system with VirD4 bound, define the biochemical requirements for complex formation and describe the protein-protein interaction network in which VirD4 is involved. We also solve the structure of this complex by negative stain electron microscopy, demonstrating that two copies of VirD4 dimers locate on both sides of the apparatus, in between the VirB4 ATPases. Given the central role of VirD4 in type IV secretion, our study provides mechanistic insights on a process that mediates the dangerous spread of antibiotic resistance genes among bacterial populations.
History
DepositionFeb 3, 2017-
Header (metadata) releaseMar 8, 2017-
Map releaseJan 17, 2018-
UpdateJul 31, 2019-
Current statusJul 31, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.39
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.39
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3585.png

Downloads & links

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Map

FileDownload / File: emd_3585.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain electron microscopy of the T4SS3-10 D4 complex enableslocalization of the VirD4 coupling protein within the complex.
Voxel sizeX=Y=Z: 1.54 Å
Density
Contour LevelBy AUTHOR: 1.39 / Movie #1: 1.39
Minimum - Maximum-2.8706295 - 14.235293
Average (Standard dev.)0.000000003300332 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-150-150-150
Dimensions300300300
Spacing300300300
CellA=B=C: 462.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.541.541.54
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z462.000462.000462.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-150-150-150
NC/NR/NS300300300
D min/max/mean-2.87114.2350.000

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Supplemental data

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Sample components

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Entire : Type IV secretion system, inner membrane complex

EntireName: Type IV secretion system, inner membrane complexSecretion
Components
  • Complex: Type IV secretion system, inner membrane complexSecretion

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Supramolecule #1: Type IV secretion system, inner membrane complex

SupramoleculeName: Type IV secretion system, inner membrane complex / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: TOP10 / Recombinant plasmid: pR388
Molecular weightTheoretical: 1.5 MDa

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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

Concentration0.03 mg/mL
BufferpH: 7.6
Details: 50mM HEPES pH=7.6, 200mM sodium acetate, 0.1% digitonin, 0.05mM TDAO
StainingType: NEGATIVE / Material: 2 % uranyl acetate / Details: Staining for 3 min
GridModel: type B 400, Agar Scientific / Material: COPPER / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 4.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 41500 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm
Image recordingFilm or detector model: DIRECT ELECTRON DE-20 (5k x 3k) / Detector mode: INTEGRATING / Digitization - Frames/image: 2-19 / Average exposure time: 2.5 sec. / Average electron dose: 30.0 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 14918
CTF correctionSoftware - Name: CTFFIND (ver. 3)
Startup modelType of model: EMDB MAP
EMDB ID:

2567

Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: IMAGIC (ver. 5)
Final 3D classificationNumber classes: 2000 / Avg.num./class: 8 / Software - Name: IMAGIC (ver. 5)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: IMAGIC (ver. 5)
Final reconstructionNumber classes used: 700 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 28.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMAGIC (ver. 5) / Number images used: 2800

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