EMDB-3649: Unraveling the self-assembly of Pseudomonas aeruginosa XcpQ secre...

Basic information

• Entry: Database: EMDB / ID: EMD-3649
• Title: Unraveling the self-assembly of Pseudomonas aeruginosa XcpQ secretin periplasmic domain provides new molecular insights into T2SS secreton architecture & dynamics

Sample

• Complex: The structure of the secretin N-terminal domain S210C mutant from Pseudomonas aeruginosa.
  • Protein or peptide: Secretin N domain S210C mutant

Function / homology

Function:

protein secretion by the type II secretion system / type II protein secretion system complex / protein secretion / cell outer membrane / identical protein binding

Domain/homology:

Type II secretion system protein GspD / : / GspD-like, N0 domain / 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

Component:

Type II secretion system protein D / Secretin XcpQ

• Biological species: Pseudomonas aeruginosa (bacteria)
• Method: single particle reconstruction / negative staining / Resolution: 24.8 Å
• Authors: Douzi B / Trinh N / Ball G / Desmyter A / Michel-Souzy S / Barbier P / Kosta A / Durand E / Cambillau C / Roussel A / Vouloux R
• Citation: Journal: mBio / Year: 2017; Title: Unraveling the Self-Assembly of the XcpQ Secretin Periplasmic Domain Provides New Molecular Insights into Type II Secretion System Secreton Architecture and Dynamics.; Authors: Badreddine Douzi / Nhung T T Trinh / Sandra Michel-Souzy / Aline Desmyter / Geneviève Ball / Pascale Barbier / Artemis Kosta / Eric Durand / Katrina T Forest / Christian Cambillau / Alain Roussel / Romé Voulhoux / ; Abstract: The type II secretion system (T2SS) releases large folded exoproteins across the envelope of many Gram-negative pathogens. This secretion process therefore requires specific gating, interacting, and dynamics properties mainly operated by a bipartite outer membrane channel called secretin. We have a good understanding of the structure-function relationship of the pore-forming C-terminal domain of secretins. In contrast, the high flexibility of their periplasmic N-terminal domain has been an obstacle in obtaining the detailed structural information required to uncover its molecular function. In , the Xcp T2SS plays an important role in bacterial virulence by its capacity to deliver a large panel of toxins and degradative enzymes into the surrounding environment. Here, we revealed that the N-terminal domain of XcpQ secretin spontaneously self-assembled into a hexamer of dimers independently of its C-terminal domain. Furthermore, and by using multidisciplinary approaches, we elucidate the structural organization of the XcpQ N domain and demonstrate that secretin flexibility at interdimer interfaces is mandatory for its function. Bacterial secretins are large homooligomeric proteins constituting the outer membrane pore-forming element of several envelope-embedded nanomachines essential in bacterial survival and pathogenicity. They comprise a well-defined membrane-embedded C-terminal domain and a modular periplasmic N-terminal domain involved in substrate recruitment and connection with inner membrane components. We are studying the XcpQ secretin of the T2SS present in the pathogenic bacterium Our data highlight the ability of the XcpQ N-terminal domain to spontaneously oligomerize into a hexamer of dimers. Further experiments revealed that this domain adopts different conformations essential for the T2SS secretion process. These findings provide new insights into the functional understanding of bacterial T2SS secretins.

History

Deposition	Mar 23, 2017	-
Header (metadata) release	Jun 14, 2017	-
Map release	Dec 20, 2017	-
Update	Jun 13, 2018	-
Current status	Jun 13, 2018	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_3649.map.gz / Format: CCP4 / Size: 2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Voxel size: X=Y=Z: 4.4 Å

Density

Contour Level	By AUTHOR: 1.33 / Movie #1: 1.33
Minimum - Maximum	-1.6932919 - 5.0239944
Average (Standard dev.)	0.022972757 (±0.22302715)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin -40 -40 -40 Dimensions 80 80 80 Spacing 80 80 80
Cell	A=B=C: 352.0 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	4.4	4.4	4.4
M x/y/z	80	80	80
origin x/y/z	0.000	0.000	0.000
length x/y/z	352.000	352.000	352.000
α/β/γ	90.000	90.000	90.000
MAP C/R/S	1	2	3
start NC/NR/NS	-40	-40	-40
NC/NR/NS	80	80	80
D min/max/mean	-1.693	5.024	0.023

Supplemental data

Sample components

Entire : The structure of the secretin N-terminal domain S210C mutant from...

• Entire: Name: The structure of the secretin N-terminal domain S210C mutant from Pseudomonas aeruginosa.

Components

• Complex: The structure of the secretin N-terminal domain S210C mutant from Pseudomonas aeruginosa.
  • Protein or peptide: Secretin N domain S210C mutant

Supramolecule #1: The structure of the secretin N-terminal domain S210C mutant from...

• Supramolecule: Name: The structure of the secretin N-terminal domain S210C mutant from Pseudomonas aeruginosa.; type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
• Source (natural): Organism: Pseudomonas aeruginosa (bacteria)
• Recombinant expression: Organism: Escherichia coli BL21(DE3) (bacteria)

Macromolecule #1: Secretin N domain S210C mutant

• Macromolecule: Name: Secretin N domain S210C mutant / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
• Source (natural): Organism: Pseudomonas aeruginosa (bacteria)
• Recombinant expression: Organism: Escherichia coli BL21(DE3) (bacteria)

Sequence

String:

E N S G G N A F V P A G N Q Q E A H W T I N L K D A D I R E F I D Q I S E I T G E T F V V D P R V K G Q V S V V S K A Q L S L S E V Y Q L F L S V M S T H G F T V V A Q G D Q A R I V P N A E A K T E A G G G Q S A P D R L E T R V I Q V Q Q S P V S E L I P L I R P L V P Q Y G H L A A V P S A N A L I I S D R S A N I A R I E D V I R Q L D Q K G S H D Y C V I N L R Y G W V M D A A E V L N N A M S R G Q A K G A A G A Q V I A D A R T N R L I I L G P P Q A R A K L V Q L A Q S L D T P

Experimental details

Structure determination

• Method: negative staining
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 1 mg/mL

Buffer

pH: 8 / Component:

Concentration	Name
50.0 mM	Tris-Hcl
150.0 mM	NaCl

• Staining: Type: NEGATIVE / Material: Uranyl acetate; Details: The protein was coated on carbon grid for 3 min. The grid were then washed using drop method 3 times and then incubated with 2% Uranyl acetate for 3 min.
• Grid: Model: electron microscopy Sciences / Material: NICKEL / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 400.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
• Details: this sample was monodisperse

Electron microscopy

• Microscope: FEI TECNAI 20
• Image recording: Film or detector model: FEI EAGLE (2k x 2k) / Number grids imaged: 3 / Number real images: 103 / Average electron dose: 2.0 e/Ų
• Electron beam: Acceleration voltage: 200 kV / Electron source: LAB6
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm

Image processing

• Particle selection: Number selected: 3600 ; Details: negative monitor contrast facilitated particle manual picking using EMAN2
• CTF correction: Software - Name: EMAN (ver. 2.12)
• Startup model: Type of model: OTHER; Details: Initial model created by EMAN2 based on the best 2D classes
• Final reconstruction: Number classes used: 25 / Applied symmetry - Point group: C₆ (6 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 24.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: EMAN (ver. 2.12) / Number images used: 3600
• Initial angle assignment: Type: NOT APPLICABLE
• Final angle assignment: Type: NOT APPLICABLE