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- EMDB-0327: Vibrio vulnificus EpsD -

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Basic information

Entry
Database: EMDB / ID: EMD-0327
TitleVibrio vulnificus EpsD
Map dataEpsD 3D reconstruction
Sample
  • Complex: EpsD
    • Protein or peptide: General 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 / membrane => GO:0016020 / cell outer membrane
Similarity search - Function
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 / NolW-like superfamily / Bacterial type II/III secretion system short domain / Type II/III secretion system / Bacterial type II and III secretion system protein
Similarity search - Domain/homology
General secretion pathway protein GspD
Similarity search - Component
Biological speciesVibrio vulnificus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsContreras-Martel C / Farias Estrozi L
Funding support France, Canada, 3 items
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, 2018-
Header (metadata) releaseApr 10, 2019-
Map releaseApr 10, 2019-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.063795
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.063795
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6i1y
  • Surface level: 0.063795
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0327.png

Downloads & links

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Map

FileDownload / File: emd_0327.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEpsD 3D reconstruction
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.21 Å/pix.
x 250 pix.
= 302.5 Å		1.21 Å/pix.
x 250 pix.
= 302.5 Å		1.21 Å/pix.
x 250 pix.
= 302.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.21 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.063795 / Movie #1: 0.063795
Minimum - Maximum-0.16631979 - 0.31914046
Average (Standard dev.)0.0019775715 (±0.012758805)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-125-125-125
Dimensions250250250
Spacing250250250
CellA=B=C: 302.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.211.211.21
M x/y/z250250250
origin x/y/z0.0000.0000.000
length x/y/z302.500302.500302.500
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-125-125-125
NC/NR/NS250250250
D min/max/mean-0.1660.3190.002

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Supplemental data

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Mask #1

Fileemd_0327_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : EpsD

EntireName: EpsD
Components
  • Complex: EpsD
    • Protein or peptide: General secretion pathway protein GspD

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Supramolecule #1: EpsD

SupramoleculeName: EpsD / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Vibrio vulnificus (bacteria)
Molecular weightTheoretical: 1 MDa

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Macromolecule #1: General secretion pathway protein GspD

MacromoleculeName: General secretion pathway protein GspD / type: protein_or_peptide / ID: 1 / Number of copies: 15 / Enantiomer: LEVO
Source (natural)Organism: Vibrio vulnificus (bacteria)
Molecular weightTheoretical: 60.124922 KDa
Recombinant expressionOrganism: Cell-free gateway cloning vector N-term 8xHis pCellFree_G01 (others)
SequenceString: DNVITRVVAV RNVSVRELSP LLRQLIDNAG AGNVVHYDPA NIILITGRAA VVNRLAEIIK RVDQAGNREI EVVELGNASA AEMVRIVDA LNRTTDAKNT PEFLQPKLVA DERTNSILIS GDPKVRDRLK RLIRQLDVEM ASKGNNRVVY LKYAKAEDLV D VLKGVSDN ...String:
DNVITRVVAV RNVSVRELSP LLRQLIDNAG AGNVVHYDPA NIILITGRAA VVNRLAEIIK RVDQAGNREI EVVELGNASA AEMVRIVDA LNRTTDAKNT PEFLQPKLVA DERTNSILIS GDPKVRDRLK RLIRQLDVEM ASKGNNRVVY LKYAKAEDLV D VLKGVSDN LQAEKNSGQK GASSQRNDVV IAAHQGTNSL VLTAPPDIML ALQEVITQLD IRRAQVLIEA LIVEMAEGDG VN LGVQWGN LETGAVIQYS NTGTPIGKVM VGLEEAKDKT VTDSRWNSDT DKYEPYSRTE AGDYSTLAAA LAGVNGAAMS LVM GDWTAL ISAVSSDSNS NILSSPSITV MDNGEASFIV GEEVPVITGS TAGSNNDNPF QTVDRKEVGI KLKVVPQINE GDSV QLNIE QEVSNVLGAN GAVDVRFAKR QLNTSVIVQD GQMLVLGGLI DERALESESK VPLLGDIPIL GHLFKSTNTQ VEKKN LMVF IKPTIIRDGM TADGITQRKY NYIRAEQLYK AEQGLKLMDD GHIPVLPKFG EDKRHPAEIQ AFIDQMEQQ

UniProtKB: General secretion pathway protein GspD

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration10 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was monodisperse

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Electron microscopy

MicroscopeFEI POLARA 300
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 2021 / Average exposure time: 1.0 sec. / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 41322 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

DetailsImage processing performed with motioncor2, Gctf, EMAN and Relion.
Particle selectionNumber selected: 125884
Startup modelType of model: OTHER / Details: Ab-initio model by using: http://rico.ibs.fr
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C15 (15 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 46126
Initial angle assignmentType: OTHER / Software - Name: RELION (ver. 2.1) / Details: Ab-initio model by using: http://rico.ibs.fr
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 2.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5wq8


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsCoot and buccaneer for model building and refmac5 for refinement (CCPEM-1.1.0 suite).
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 146 / Target criteria: Cross-correlation coefficient
Output model

PDB-6i1y:
Vibrio vulnificus EpsD

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