+Open data
-Basic information
Entry | Database: PDB / ID: 5wda | ||||||||||||
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Title | Structure of the PulG pseudopilus | ||||||||||||
Components | General secretion pathway protein G | ||||||||||||
Keywords | PROTEIN TRANSPORT / helical polymer / bacterial secretion / cryo-EM | ||||||||||||
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 | ||||||||||||
Biological species | Klebsiella oxytoca (bacteria) | ||||||||||||
Method | ELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 5 Å | ||||||||||||
Authors | Lopez-Castilla, A. / Thomassin, J.L. / Bardiaux, B. / Zheng, W. / Nivaskumar, M. / Yu, X. / Nilges, M. / Egelman, E.H. / Izadi-Pruneyre, N. / Francetic, O. | ||||||||||||
Funding support | United States, France, European Union, 3items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 5wda.cif.gz | 999.8 KB | Display | PDBx/mmCIF format |
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PDB format | pdb5wda.ent.gz | 873.9 KB | Display | PDB format |
PDBx/mmJSON format | 5wda.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 5wda_validation.pdf.gz | 862.5 KB | Display | wwPDB validaton report |
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Full document | 5wda_full_validation.pdf.gz | 897.1 KB | Display | |
Data in XML | 5wda_validation.xml.gz | 60.9 KB | Display | |
Data in CIF | 5wda_validation.cif.gz | 103 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/wd/5wda ftp://data.pdbj.org/pub/pdb/validation_reports/wd/5wda | HTTPS FTP |
-Related structure data
Related structure data | 8812MC 5o2yC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | Mass: 14525.482 Da / Num. of mol.: 25 / Fragment: UNP residues 7-140 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Klebsiella oxytoca (bacteria) / Gene: pulG, AB185_31145, SAMEA2273639_02747 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0G3SCW3 #2: Chemical | ChemComp-CA / |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: FILAMENT / 3D reconstruction method: helical reconstruction |
-Sample preparation
Component | Name: PulG pseudopilus / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT |
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Molecular weight | Experimental value: NO |
Source (natural) | Organism: Klebsiella oxytoca (bacteria) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 7.5 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD |
Image recording | Electron dose: 20 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1819 |
-Processing
Software | Name: PHENIX / Version: 1.11.1_2575: / Classification: refinement | ||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Helical symmerty | Angular rotation/subunit: 83.2 ° / Axial rise/subunit: 10.2 Å / Axial symmetry: C1 | ||||||||||||||||||||||||
3D reconstruction | Resolution: 5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 85619 / Algorithm: BACK PROJECTION / Symmetry type: HELICAL | ||||||||||||||||||||||||
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