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- PDB-5wda: Structure of the PulG pseudopilus -

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Basic information

Entry
Database: PDB / ID: 5wda
TitleStructure of the PulG pseudopilus
ComponentsGeneral secretion pathway protein G
KeywordsPROTEIN 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
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)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 5 Å
AuthorsLopez-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
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, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 25, 2017Provider: repository / Type: Initial release
Revision 1.1Dec 13, 2017Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support
Item: _pdbx_audit_support.country / _pdbx_audit_support.funding_organization

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Structure visualization

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Assembly

Deposited unit
A: General secretion pathway protein G
B: General secretion pathway protein G
C: General secretion pathway protein G
D: General secretion pathway protein G
E: General secretion pathway protein G
F: General secretion pathway protein G
G: General secretion pathway protein G
H: General secretion pathway protein G
I: General secretion pathway protein G
J: General secretion pathway protein G
K: General secretion pathway protein G
L: General secretion pathway protein G
M: General secretion pathway protein G
N: General secretion pathway protein G
O: General secretion pathway protein G
P: General secretion pathway protein G
Q: General secretion pathway protein G
R: General secretion pathway protein G
S: General secretion pathway protein G
T: General secretion pathway protein G
U: General secretion pathway protein G
V: General secretion pathway protein G
W: General secretion pathway protein G
X: General secretion pathway protein G
Y: General secretion pathway protein G
hetero molecules


Theoretical massNumber of molelcules
Total (without water)364,13950
Polymers363,13725
Non-polymers1,00225
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area89630 Å2
ΔGint-692 kcal/mol
Surface area128840 Å2

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Components

#1: Protein ...
General secretion pathway protein G / General secretion pathway protein GspG


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 / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 25 / Source method: obtained synthetically / Formula: Ca

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: PulG pseudopilus / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Klebsiella oxytoca (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron 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

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameCategory
4CTFFIND3CTF correction
13IHRSR3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 83.2 ° / Axial rise/subunit: 10.2 Å / Axial symmetry: C1
3D reconstructionResolution: 5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 85619 / Algorithm: BACK PROJECTION / Symmetry type: HELICAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01249625
ELECTRON MICROSCOPYf_angle_d1.43190250
ELECTRON MICROSCOPYf_dihedral_angle_d3.06419400
ELECTRON MICROSCOPYf_chiral_restr0.0683900
ELECTRON MICROSCOPYf_plane_restr0.00811075

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