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- PDB-5o2y: NMR structure of the calcium bound form of PulG, major pseudopili... -

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Basic information

Entry
Database: PDB / ID: 5o2y
TitleNMR structure of the calcium bound form of PulG, major pseudopilin from Klebsiella oxytoca T2SS
ComponentsGeneral secretion pathway protein G
KeywordsPROTEIN TRANSPORT / Klebsiella oxytoca T2SS / major pseudopilin / calcium
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 / Glycoprotein, Type 4 Pilin / Bacterial general secretion pathway protein G-type pilin / Glycoprotein, Type 4 Pilin / Prokaryotic N-terminal methylation site. / Prokaryotic N-terminal methylation motif / Prokaryotic N-terminal methylation site / Pilin-like ...Type II secretion system protein GspG / Type II secretion system protein GspG, C-terminal / Type II secretion system (T2SS), protein G / Glycoprotein, Type 4 Pilin / Bacterial general secretion pathway protein G-type pilin / Glycoprotein, Type 4 Pilin / Prokaryotic N-terminal methylation site. / Prokaryotic N-terminal methylation motif / Prokaryotic N-terminal methylation site / Pilin-like / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
Type II secretion system core protein G
Similarity search - Component
Biological speciesKlebsiella oxytoca (bacteria)
MethodSOLUTION NMR / simulated annealing
AuthorsLopez-Castilla, A. / Bardiaux, B. / Vitorge, B. / Thomassin, J.-L. / Zheng, W. / Yu, X. / Egelman, E.H. / Nilges, M. / Francetic, O. / Izadi-Pruneyre, N.
Funding support France, 1items
OrganizationGrant numberCountry
French National Research AgencyANR-14-CE09-0004NR France
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
DepositionMay 23, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 18, 2017Provider: repository / Type: Initial release
Revision 1.1Dec 6, 2017Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name
Revision 1.2May 8, 2019Group: Data collection / Category: pdbx_nmr_software / Item: _pdbx_nmr_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: General secretion pathway protein G
hetero molecules


Theoretical massNumber of molelcules
Total (without water)12,9682
Polymers12,9281
Non-polymers401
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area7530 Å2
MethodPISA
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)15 / 200structures with the least restraint violations
RepresentativeModel #1lowest energy

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Components

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


Mass: 12928.186 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella oxytoca (bacteria) / Gene: pulG, AB185_31145, SAMEA2273639_02747 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A0G3SCW3
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
111isotropic12D 1H-15N HSQC
121isotropic13D HNCO
131isotropic1HN(CA)CO
141isotropic13D HN(CA)CB
151isotropic13D CBCA(CO)NH
161isotropic13D (H)CCH-TOCSY
171isotropic13D H(CCO)NH
181isotropic23D 1H-15N NOESY
191isotropic23D 1H-13C NOESY
1101isotropic12D 1H-13C HSQC
1111isotropic13D CC(CO)NH
1121isotropic12D (HB)CB(CGCD)HD
1131isotropic12D (HB)CB(CGCDCE)HE

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Sample preparation

DetailsType: solution
Contents: 0.5 mM [U-99% 13C; U-99% 15N] PulG, 50 mM non-labeled HEPES, 50 mM non-labeled sodium chloride, 1 mM non-labeled CaCl2, 90% H2O/10% D2O
Label: 15N_13C / Solvent system: 90% H2O/10% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
0.5 mMPulG[U-99% 13C; U-99% 15N]1
50 mMHEPESnon-labeled1
50 mMsodium chloridenon-labeled1
1 mMCaCl2non-labeled1
Sample conditionsIonic strength: 50 mM / Label: conditions_1 / pH: 7 / Pressure: 1 atm / Temperature: 298 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Varian INOVAVarianINOVA6001
Bruker AvanceIIIBrukerAvanceIII9502

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Processing

NMR software
NameDeveloperClassification
VNMRVariancollection
NMRPipeDelaglio, Grzesiek, Vuister, Zhu, Pfeifer and Baxprocessing
CcpNmr AnalysisCCPNchemical shift assignment
CcpNmr AnalysisCCPNpeak picking
TopSpinBruker Biospincollection
TopSpinBruker Biospinprocessing
ARIALinge, O'Donoghue and Nilgesstructure calculation
RefinementMethod: simulated annealing / Software ordinal: 7
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with the least restraint violations
Conformers calculated total number: 200 / Conformers submitted total number: 15

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