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- EMDB-3112: Negative stain EM structure of the Tse6-Tsi6-VgrG1-EF-Tu complex ... -

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Basic information

Entry
Database: EMDB / ID: EMD-3112
TitleNegative stain EM structure of the Tse6-Tsi6-VgrG1-EF-Tu complex in detergent
Map dataReconstruction of Tse6-Tsi6-VgrG1-EF-Tu complex in detergent
Sample
  • Sample: Tse6-Tsi6-VgrG1-EF-Tu complex in detergent
  • Protein or peptide: Valine-glycine repeat protein 1
  • Protein or peptide: Elongation factor Tu 1EF-Tu
  • Protein or peptide: Type VI secretion immunity 6Type VI secretion system
  • Protein or peptide: Type VI secretion exported 6Type VI secretion system
KeywordsType VI secretion system / bacterial competition / polymicrobial / effector / glycohydrolase
Function / homology
Function and homology information


type VI protein secretion system complex / protein secretion by the type VI secretion system / : / NAD+ glycohydrolase / toxin sequestering activity / guanyl-nucleotide exchange factor complex / NADP+ nucleosidase activity / guanosine tetraphosphate binding / translational elongation / translation elongation factor activity ...type VI protein secretion system complex / protein secretion by the type VI secretion system / : / NAD+ glycohydrolase / toxin sequestering activity / guanyl-nucleotide exchange factor complex / NADP+ nucleosidase activity / guanosine tetraphosphate binding / translational elongation / translation elongation factor activity / response to antibiotic / GTPase activity / GTP binding / RNA binding / extracellular region / membrane / plasma membrane / cytoplasm
Similarity search - Function
Tsi6 / Tsi6 / Bacterial toxin 46 / Bacterial toxin 46 / PAAR motif / Type VI secretion system, RhsGE-associated Vgr family subset / PAAR motif / Phage tail baseplate hub (GPD) / Type VI secretion system, RhsGE-associated Vgr protein / Gp5/Type VI secretion system Vgr protein, OB-fold domain ...Tsi6 / Tsi6 / Bacterial toxin 46 / Bacterial toxin 46 / PAAR motif / Type VI secretion system, RhsGE-associated Vgr family subset / PAAR motif / Phage tail baseplate hub (GPD) / Type VI secretion system, RhsGE-associated Vgr protein / Gp5/Type VI secretion system Vgr protein, OB-fold domain / Type VI secretion system/phage-baseplate injector OB domain / Vgr protein, OB-fold domain superfamily / Translation elongation factor EFTu/EF1A, bacterial/organelle / Elongation factor Tu, domain 2 / Elongation factor Tu (EF-Tu), GTP-binding domain / Translation elongation factor EFTu/EF1A, C-terminal / Elongation factor Tu C-terminal domain / Translation elongation factor EF1A/initiation factor IF2gamma, C-terminal / Tr-type G domain, conserved site / Translational (tr)-type guanine nucleotide-binding (G) domain signature. / Translation elongation factor EFTu-like, domain 2 / Elongation factor Tu domain 2 / Translational (tr)-type GTP-binding domain / Elongation factor Tu GTP binding domain / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / Small GTP-binding protein domain / Translation protein, beta-barrel domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Elongation factor Tu 1 / NAD(P)(+) glycohydrolase toxin Tse6 / Immune protein Tsi6 / Type VI secretion system spike protein VgrG1a
Similarity search - Component
Biological speciesPseudomonas aeruginosa PAO1 (bacteria) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / negative staining / Resolution: 19.4 Å
AuthorsWhitney J / Quentin D / Sawai S / LeRoux M / Harding B / Ledvina H / Tran B / Robinson H / Goo YA / Goodlett D ...Whitney J / Quentin D / Sawai S / LeRoux M / Harding B / Ledvina H / Tran B / Robinson H / Goo YA / Goodlett D / Raunser S / Mougous J
CitationJournal: Cell / Year: 2015
Title: An interbacterial NAD(P)(+) glycohydrolase toxin requires elongation factor Tu for delivery to target cells.
Authors: John C Whitney / Dennis Quentin / Shin Sawai / Michele LeRoux / Brittany N Harding / Hannah E Ledvina / Bao Q Tran / Howard Robinson / Young Ah Goo / David R Goodlett / Stefan Raunser / Joseph D Mougous /
Abstract: Type VI secretion (T6S) influences the composition of microbial communities by catalyzing the delivery of toxins between adjacent bacterial cells. Here, we demonstrate that a T6S integral membrane ...Type VI secretion (T6S) influences the composition of microbial communities by catalyzing the delivery of toxins between adjacent bacterial cells. Here, we demonstrate that a T6S integral membrane toxin from Pseudomonas aeruginosa, Tse6, acts on target cells by degrading the universally essential dinucleotides NAD(+) and NADP(+). Structural analyses of Tse6 show that it resembles mono-ADP-ribosyltransferase proteins, such as diphtheria toxin, with the exception of a unique loop that both excludes proteinaceous ADP-ribose acceptors and contributes to hydrolysis. We find that entry of Tse6 into target cells requires its binding to an essential housekeeping protein, translation elongation factor Tu (EF-Tu). These proteins participate in a larger assembly that additionally directs toxin export and provides chaperone activity. Visualization of this complex by electron microscopy defines the architecture of a toxin-loaded T6S apparatus and provides mechanistic insight into intercellular membrane protein delivery between bacteria.
History
DepositionAug 6, 2015-
Header (metadata) releaseAug 26, 2015-
Map releaseOct 21, 2015-
UpdateNov 25, 2015-
Current statusNov 25, 2015Processing 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
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  • Surface view colored by height
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3112.tif

Downloads & links

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Map

FileDownload / File: emd_3112.map.gz / Format: CCP4 / Size: 41.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of Tse6-Tsi6-VgrG1-EF-Tu complex in detergent
Voxel sizeX=Y=Z: 2.32 Å
Density
Contour LevelBy AUTHOR: 0.25 / Movie #1: 0.25
Minimum - Maximum-3.02531624 - 4.7177062
Average (Standard dev.)-0.00000439 (±0.13986629)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 519.68 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.322.322.32
M x/y/z224224224
origin x/y/z0.0000.0000.000
length x/y/z519.680519.680519.680
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS224224224
D min/max/mean-3.0254.718-0.000

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Supplemental data

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Sample components

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Entire : Tse6-Tsi6-VgrG1-EF-Tu complex in detergent

EntireName: Tse6-Tsi6-VgrG1-EF-Tu complex in detergent
Components
  • Sample: Tse6-Tsi6-VgrG1-EF-Tu complex in detergent
  • Protein or peptide: Valine-glycine repeat protein 1
  • Protein or peptide: Elongation factor Tu 1EF-Tu
  • Protein or peptide: Type VI secretion immunity 6Type VI secretion system
  • Protein or peptide: Type VI secretion exported 6Type VI secretion system

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Supramolecule #1000: Tse6-Tsi6-VgrG1-EF-Tu complex in detergent

SupramoleculeName: Tse6-Tsi6-VgrG1-EF-Tu complex in detergent / type: sample / ID: 1000
Oligomeric state: Trimer of VgrG1 binds to monomers of Tse6, Tsi6 and EF-Tu
Number unique components: 4
Molecular weightTheoretical: 331 KDa

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Macromolecule #1: Valine-glycine repeat protein 1

MacromoleculeName: Valine-glycine repeat protein 1 / type: protein_or_peptide / ID: 1 / Name.synonym: VgrG1 / Number of copies: 3 / Oligomeric state: Trimer / Recombinant expression: Yes
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)
Molecular weightTheoretical: 72 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: Type VI secretion system spike protein VgrG1a

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Macromolecule #2: Elongation factor Tu 1

MacromoleculeName: Elongation factor Tu 1 / type: protein_or_peptide / ID: 2 / Name.synonym: Ef-Tu / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 43 KDa
SequenceUniProtKB: Elongation factor Tu 1

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Macromolecule #3: Type VI secretion immunity 6

MacromoleculeName: Type VI secretion immunity 6 / type: protein_or_peptide / ID: 3 / Name.synonym: Tsi6 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)
Molecular weightTheoretical: 11 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: Immune protein Tsi6

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Macromolecule #4: Type VI secretion exported 6

MacromoleculeName: Type VI secretion exported 6 / type: protein_or_peptide / ID: 4 / Name.synonym: Tse6 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria) / synonym: Pseudomonas aeruginosa
Molecular weightTheoretical: 45 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: NAD(P)(+) glycohydrolase toxin Tse6

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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

Concentration0.015 mg/mL
BufferpH: 8 / Details: 150 mM NaCl, 20 mM Tris, 0,03 % DDM
StainingType: NEGATIVE
Details: Grids with adsorbed Protein floated on 0.75% Uranyl Formate for 45 sec.
GridDetails: glow discharged 400 mesh copper grids with thin carbon support
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeJEOL 1400
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 67210 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 3.4 mm / Nominal defocus max: 0.002 µm / Nominal defocus min: 0.0012 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
DateMar 30, 2015
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 252 / Average electron dose: 10 e/Å2

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 19.4 Å / Resolution method: OTHER / Software - Name: SPARX / Number images used: 4684

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