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- EMDB-8812: Structure of the PulG pseudopilus -

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Basic information

Entry
Database: EMDB / ID: EMD-8812
TitleStructure of the PulG pseudopilus
Map datamap of PulG filament
Sample
  • Organelle or cellular component: PulG pseudopilus
    • Protein or peptide: General secretion pathway protein G
    • Ligand: CALCIUM ION
Keywordshelical polymer / bacterial secretion / cryo-EM / PROTEIN TRANSPORT
Function / homology
Function and homology information


protein secretion by the type II secretion system / type II protein secretion system complex / membrane => GO:0016020 / plasma membrane
Similarity search - Function
Type II secretion system protein GspG / Type II secretion system protein GspG, C-terminal / Type II secretion system (T2SS), protein G / Bacterial general secretion pathway protein G-type pilin / Prokaryotic N-terminal methylation site. / Prokaryotic N-terminal methylation motif / Prokaryotic N-terminal methylation site / Pilin-like
Similarity search - Domain/homology
Type II secretion system core protein G
Similarity search - Component
Biological speciesKlebsiella oxytoca (bacteria)
Methodhelical reconstruction / cryo EM / Resolution: 5.0 Å
AuthorsLopez-Castilla A / Thomassin JL
Funding support United States, France, European Union, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM122510 United States
Agence Nationale de la RechercheANR-14-CE09-0004 France
European Union (EU)FP7-IDEAS-ERC 294809European Union
CitationJournal: Nat Microbiol / Year: 2017
Title: Structure of the calcium-dependent type 2 secretion pseudopilus.
Authors: Aracelys López-Castilla / Jenny-Lee Thomassin / Benjamin Bardiaux / Weili Zheng / Mangayarkarasi Nivaskumar / Xiong Yu / Michael Nilges / Edward H Egelman / Nadia Izadi-Pruneyre / Olivera Francetic /
Abstract: Many Gram-negative bacteria use type 2 secretion systems (T2SSs) to secrete proteins involved in virulence and adaptation. Transport of folded proteins via T2SS nanomachines requires the assembly of ...Many Gram-negative bacteria use type 2 secretion systems (T2SSs) to secrete proteins involved in virulence and adaptation. Transport of folded proteins via T2SS nanomachines requires the assembly of inner membrane-anchored fibres called pseudopili. Although efficient pseudopilus assembly is essential for protein secretion, structure-based functional analyses are required to unravel the mechanistic link between these processes. Here, we report an atomic model for a T2SS pseudopilus from Klebsiella oxytoca, obtained by fitting the NMR structure of its calcium-bound subunit PulG into the ~5-Å-resolution cryo-electron microscopy reconstruction of assembled fibres. This structure reveals the comprehensive network of inter-subunit contacts and unexpected features, including a disordered central region of the PulG helical stem, and highly flexible C-terminal residues on the fibre surface. NMR, mutagenesis and functional analyses highlight the key role of calcium in PulG folding and stability. Fibre disassembly in the absence of calcium provides a basis for pseudopilus length control, essential for protein secretion, and supports the Archimedes screw model for the type 2 secretion mechanism.
History
DepositionJul 4, 2017-
Header (metadata) releaseAug 9, 2017-
Map releaseOct 25, 2017-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.056
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.056
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5wda
  • Surface level: 0.056
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5wda
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8812.png

Downloads & links

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Map

FileDownload / File: emd_8812.map.gz / Format: CCP4 / Size: 8.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmap of PulG filament
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 300 pix.
= 315. Å		1.05 Å/pix.
x 84 pix.
= 88.2 Å		1.05 Å/pix.
x 84 pix.
= 88.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 1.05 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.056 / Movie #1: 0.056
Minimum - Maximum-0.044437073 - 0.11336735
Average (Standard dev.)0.012011195 (±0.02283012)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-42-42-150
Dimensions8484300
Spacing8484300
CellA: 88.2 Å / B: 88.2 Å / C: 315.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z8484300
origin x/y/z0.0000.0000.000
length x/y/z88.20088.200315.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-42-42-150
NC/NR/NS8484300
D min/max/mean-0.0440.1130.012

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Supplemental data

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Sample components

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Entire : PulG pseudopilus

EntireName: PulG pseudopilus
Components
  • Organelle or cellular component: PulG pseudopilus
    • Protein or peptide: General secretion pathway protein G
    • Ligand: CALCIUM ION

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Supramolecule #1: PulG pseudopilus

SupramoleculeName: PulG pseudopilus / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Klebsiella oxytoca (bacteria)

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Macromolecule #1: General secretion pathway protein G

MacromoleculeName: General secretion pathway protein G / type: protein_or_peptide / ID: 1 / Number of copies: 25 / Enantiomer: LEVO
Source (natural)Organism: Klebsiella oxytoca (bacteria)
Molecular weightTheoretical: 14.525482 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
(MEA)TLLEIMVVI VILGVLASLV VPNLMGNKEK ADRQKVVSDL VALEGALDMY KLDNSRYPTT EQGLQALVSA PSAEPH ARN YPEGGYIRRL PQDPWGSDYQ LLSPGQCGQV DIFSLGPDGV PESNDDIGNC TIGKK

UniProtKB: Type II secretion system core protein G

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Macromolecule #2: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 25 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 1 / Number real images: 1819 / Average electron dose: 20.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 10.2 Å
Applied symmetry - Helical parameters - Δ&Phi: 83.2 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 5.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: IHRSR / Number images used: 85619
Startup modelType of model: OTHER / Details: solid cylinder
Final angle assignmentType: NOT APPLICABLE

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