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- PDB-6i1x: Aeromonas hydrophila ExeD -

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Basic information

Entry
Database: PDB / ID: 6i1x
TitleAeromonas hydrophila ExeD
ComponentsType II secretion system protein DType II secretion system
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
Biological speciesAeromonas hydrophila (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 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 1.1May 8, 2019Group: Advisory / Data collection / Derived calculations
Category: em_admin / pdbx_database_proc ...em_admin / pdbx_database_proc / pdbx_validate_close_contact / struct_conn / struct_conn_type
Item: _em_admin.last_update
Revision 1.2Nov 6, 2019Group: Data collection / Database references / Refinement description
Category: citation / citation_author / em_3d_fitting
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 / _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:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Type II secretion system protein D
B: Type II secretion system protein D
C: Type II secretion system protein D
D: Type II secretion system protein D
E: Type II secretion system protein D
F: Type II secretion system protein D
G: Type II secretion system protein D
H: Type II secretion system protein D
I: Type II secretion system protein D
J: Type II secretion system protein D
K: Type II secretion system protein D
L: Type II secretion system protein D
M: Type II secretion system protein D
N: Type II secretion system protein D
O: Type II secretion system protein D


Theoretical massNumber of molelcules
Total (without water)834,27615
Polymers834,27615
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy, 2D class averages
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area185240 Å2
ΔGint-700 kcal/mol
Surface area290710 Å2
MethodPISA

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Components

#1: Protein
Type II secretion system protein D / Type II secretion system / T2SS protein D / General secretion pathway protein D


Mass: 55618.422 Da / Num. of mol.: 15
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aeromonas hydrophila (bacteria) / Gene: exeD
Production host: Cell-free gateway cloning vector N-term 8xHis pCellFree_G01 (others)
References: UniProt: P31780

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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: ExeD / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 1 MDa / Experimental value: NO
Source (natural)Organism: Aeromonas hydrophila (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
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
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: 2247
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: 272432
SymmetryPoint symmetry: C15 (15 fold cyclic)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 51960 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 182 / 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: 5WQ7

5wq7

RefinementResolution: 3.7→302.5 Å / Cor.coef. Fo:Fc: 0.77 / SU B: 69.389 / SU ML: 0.965 / ESU R: 0.439
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.48634 --
obs0.48634 1144565 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 402.416 Å2
Baniso -1Baniso -2Baniso -3
1--13.03 Å20.5 Å20.1 Å2
2---13.53 Å2-0.2 Å2
3---26.57 Å2
Refinement stepCycle: 1 / Total: 54915
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0070.01355485
ELECTRON MICROSCOPYr_bond_other_d00.01753205
ELECTRON MICROSCOPYr_angle_refined_deg1.4771.6475255
ELECTRON MICROSCOPYr_angle_other_deg1.341.576123285
ELECTRON MICROSCOPYr_dihedral_angle_1_deg8.64957245
ELECTRON MICROSCOPYr_dihedral_angle_2_deg30.0823.2792745
ELECTRON MICROSCOPYr_dihedral_angle_3_deg16.423159765
ELECTRON MICROSCOPYr_dihedral_angle_4_deg17.41415375
ELECTRON MICROSCOPYr_chiral_restr0.0750.27545
ELECTRON MICROSCOPYr_gen_planes_refined0.0060.0262460
ELECTRON MICROSCOPYr_gen_planes_other0.0010.0210425
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_it5.90643.89529115
ELECTRON MICROSCOPYr_mcbond_other5.90643.89529114
ELECTRON MICROSCOPYr_mcangle_it7.51865.83736315
ELECTRON MICROSCOPYr_mcangle_other7.51865.83736316
ELECTRON MICROSCOPYr_scbond_it4.40943.95326370
ELECTRON MICROSCOPYr_scbond_other4.4143.95326368
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other5.69465.90238940
ELECTRON MICROSCOPYr_long_range_B_refined5.824217118
ELECTRON MICROSCOPYr_long_range_B_other5.824217113
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.7→3.796 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork10.266 85228 -
obs--100 %

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