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- PDB-6i1y: Vibrio vulnificus EpsD -

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Basic information

Entry
Database: PDB / ID: 6i1y
TitleVibrio vulnificus EpsD
ComponentsGeneral secretion pathway protein GspD
KeywordsPROTEIN TRANSPORT
Function / homology
Function and homology information


protein secretion by the type II secretion system / type II protein secretion system complex / cell outer membrane / membrane => GO:0016020
Similarity search - Function
Ribosomal Protein S8; Chain: A, domain 1 - #120 / Type II secretion system protein GspD / GspD/PilQ family / Bacterial type II secretion system protein D signature. / Type II secretion system protein GspD, conserved site / NolW-like / Bacterial type II/III secretion system short domain / NolW-like superfamily / Type II/III secretion system / Bacterial type II and III secretion system protein ...Ribosomal Protein S8; Chain: A, domain 1 - #120 / Type II secretion system protein GspD / GspD/PilQ family / Bacterial type II secretion system protein D signature. / Type II secretion system protein GspD, conserved site / NolW-like / Bacterial type II/III secretion system short domain / NolW-like superfamily / Type II/III secretion system / Bacterial type II and III secretion system protein / Ribosomal Protein S8; Chain: A, domain 1 / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
General secretion pathway protein GspD
Similarity search - Component
Biological speciesVibrio vulnificus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsContreras-Martel, C. / Farias Estrozi, L.
Funding support France, Canada, 3items
OrganizationGrant numberCountry
French National Research AgencyANR-10-INSB-05-02 France
French National Research AgencyANR-10-LABX-49-01 France
Natural Sciences and Engineering Research Council (Canada) Canada
CitationJournal: PLoS Pathog / Year: 2019
Title: Structure and assembly of pilotin-dependent and -independent secretins of the type II secretion system.
Authors: S Peter Howard / Leandro F Estrozi / Quentin Bertrand / Carlos Contreras-Martel / Timothy Strozen / Viviana Job / Alexandre Martins / Daphna Fenel / Guy Schoehn / Andréa Dessen /
Abstract: The type II secretion system (T2SS) is a cell envelope-spanning macromolecular complex that is prevalent in Gram-negative bacterial species. It serves as the predominant virulence mechanism of many ...The type II secretion system (T2SS) is a cell envelope-spanning macromolecular complex that is prevalent in Gram-negative bacterial species. It serves as the predominant virulence mechanism of many bacteria including those of the emerging human pathogens Vibrio vulnificus and Aeromonas hydrophila. The system is composed of a core set of highly conserved proteins that assemble an inner membrane platform, a periplasmic pseudopilus and an outer membrane complex termed the secretin. Localization and assembly of secretins in the outer membrane requires recognition of secretin monomers by two different partner systems: an inner membrane accessory complex or a highly sequence-diverse outer membrane lipoprotein, termed the pilotin. In this study, we addressed the question of differential secretin assembly mechanisms by using cryo-electron microscopy to determine the structures of the secretins from A. hydrophila (pilotin-independent ExeD) and V. vulnificus (pilotin-dependent EpsD). These structures, at approximately 3.5 Å resolution, reveal pentadecameric stoichiometries and C-terminal regions that carry a signature motif in the case of a pilotin-dependent assembly mechanism. We solved the crystal structure of the V. vulnificus EpsS pilotin and confirmed the importance of the signature motif for pilotin-dependent secretin assembly by performing modelling with the C-terminus of EpsD. We also show that secretin assembly is essential for membrane integrity and toxin secretion in V. vulnificus and establish that EpsD requires the coordinated activity of both the accessory complex EpsAB and the pilotin EpsS for full assembly and T2SS function. In contrast, mutation of the region of the S-domain that is normally the site of pilotin interactions has little effect on assembly or function of the ExeD secretin. Since secretins are essential outer membrane channels present in a variety of secretion systems, these results provide a structural and functional basis for understanding the key assembly steps for different members of this vast pore-forming family of proteins.
History
DepositionOct 30, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 10, 2019Provider: repository / Type: Initial release
Revision 1.1Jul 31, 2019Group: Data collection / Database references / Refinement description
Category: citation / citation_author / refine
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.identifier_ORCID / _citation_author.name
Revision 1.2Nov 6, 2019Group: Data collection / Refinement description / Category: em_3d_fitting / Item: _em_3d_fitting.target_criteria
Revision 1.3Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][2] / _atom_sites.fract_transf_matrix[3][3]

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: General secretion pathway protein GspD
B: General secretion pathway protein GspD
C: General secretion pathway protein GspD
D: General secretion pathway protein GspD
E: General secretion pathway protein GspD
F: General secretion pathway protein GspD
G: General secretion pathway protein GspD
H: General secretion pathway protein GspD
I: General secretion pathway protein GspD
J: General secretion pathway protein GspD
K: General secretion pathway protein GspD
L: General secretion pathway protein GspD
M: General secretion pathway protein GspD
N: General secretion pathway protein GspD
O: General secretion pathway protein GspD


Theoretical massNumber of molelcules
Total (without water)901,87415
Polymers901,87415
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy, 2D class averages
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area188200 Å2
ΔGint-950 kcal/mol
Surface area293700 Å2
MethodPISA

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Components

#1: Protein
General secretion pathway protein GspD


Mass: 60124.922 Da / Num. of mol.: 15
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio vulnificus (bacteria) / Gene: JS86_21435
Production host: Cell-free gateway cloning vector N-term 8xHis pCellFree_G01 (others)
References: UniProt: A0A087IFK6

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: EpsD / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1 MDa / Experimental value: NO
Source (natural)Organism: Vibrio vulnificus (bacteria)
Source (recombinant)Organism: Cell-free gateway cloning vector N-term 8xHis pCellFree_G01 (others)
Plasmid: pIVEX2.4T
Buffer solutionpH: 7.5
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 41322 X / Cs: 2 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: GATAN LIQUID NITROGEN
Image recordingAverage exposure time: 1 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 2021
Image scansMovie frames/image: 40

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Processing

SoftwareName: REFMAC / Version: 5.8.0232 / Classification: refinement
EM software
IDNameVersionCategoryDetails
2LatitudeSimage acquisition
4GctfCTF correctiondetermination of CTF
5RELION2.1CTF correctionapplication of CTF
8Cootmodel fitting
10RELION2.1initial Euler assignment
11RELION2.1final Euler assignment
12RELION2.1classification
13RELION2.13D reconstruction
14REFMACmodel refinement
Image processingDetails: Image processing performed with motioncor2, Gctf, EMAN and Relion.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 125884
SymmetryPoint symmetry: C15 (15 fold cyclic)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 46126 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 146 / Protocol: OTHER / Space: REAL / Target criteria: Cross-correlation coefficient
Details: Coot and buccaneer for model building and refmac5 for refinement (CCPEM-1.1.0 suite).
Atomic model buildingPDB-ID: 5WQ8

5wq8

RefinementResolution: 3.4→302.5 Å / Cor.coef. Fo:Fc: 0.833 / SU B: 20 / SU ML: 0.301 / ESU R: 0.268
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.37052 --
obs0.37052 1474619 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 158.591 Å2
Baniso -1Baniso -2Baniso -3
1-0.24 Å2-0 Å20.03 Å2
2--0.15 Å20.01 Å2
3----0.39 Å2
Refinement stepCycle: 1 / Total: 56520
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0130.01357015
ELECTRON MICROSCOPYr_bond_other_d00.01755890
ELECTRON MICROSCOPYr_angle_refined_deg1.7581.62877175
ELECTRON MICROSCOPYr_angle_other_deg1.2451.576129390
ELECTRON MICROSCOPYr_dihedral_angle_1_deg10.18557365
ELECTRON MICROSCOPYr_dihedral_angle_2_deg27.40723.4552865
ELECTRON MICROSCOPYr_dihedral_angle_3_deg13.7011510470
ELECTRON MICROSCOPYr_dihedral_angle_4_deg11.55815390
ELECTRON MICROSCOPYr_chiral_restr0.0680.27815
ELECTRON MICROSCOPYr_gen_planes_refined0.0310.0263540
ELECTRON MICROSCOPYr_gen_planes_other0.0250.0210350
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it26.25614.1829685
ELECTRON MICROSCOPYr_mcbond_other26.25714.17929684
ELECTRON MICROSCOPYr_mcangle_it38.68421.2136975
ELECTRON MICROSCOPYr_mcangle_other38.68421.21136976
ELECTRON MICROSCOPYr_scbond_it43.05818.84927330
ELECTRON MICROSCOPYr_scbond_other43.05918.84727328
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other67.2426.33840200
ELECTRON MICROSCOPYr_long_range_B_refined76.575227768
ELECTRON MICROSCOPYr_long_range_B_other76.575227768
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.4→3.488 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.722 108939 -
obs--100 %

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