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- EMDB-8860: Cryo-EM structure of the T2SS secretin XcpQ from Pseudomonas aeru... -

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Entry
Database: EMDB / ID: 8860
TitleCryo-EM structure of the T2SS secretin XcpQ from Pseudomonas aeruginosa
Map dataT2SS secretin XcpQ from Pseudomonas aeruginosa
SampleType 2 secretion system outer membrane secretin XcpQType II secretion system
  • Type II secretion system protein DType II secretion system
Function / homologyType II secretion system GspD, conserved site / GspD/PilQ family / Type II/III secretion system / NolW-like / Type II secretion system GspD / NolW-like superfamily / Bacterial type II and III secretion system protein / Bacterial type II/III secretion system short domain / Bacterial type II secretion system protein D signature. / type II protein secretion system complex ...Type II secretion system GspD, conserved site / GspD/PilQ family / Type II/III secretion system / NolW-like / Type II secretion system GspD / NolW-like superfamily / Bacterial type II and III secretion system protein / Bacterial type II/III secretion system short domain / Bacterial type II secretion system protein D signature. / type II protein secretion system complex / protein secretion by the type II secretion system / protein transporter activity / cell outer membrane / identical protein binding / Type II secretion system protein D
Function and homology information
SourcePseudomonas aeruginosa PAO1 (bacteria) / Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) (bacteria)
Methodsingle particle reconstruction / cryo EM / 3.57 Å resolution
AuthorsHay ID / Belousoff MJ / Lithgow TJ
CitationJournal: MBio / Year: 2017
Title: Structural Basis of Type 2 Secretion System Engagement between the Inner and Outer Bacterial Membranes.
Authors: Iain D Hay / Matthew J Belousoff / Trevor Lithgow
Abstract: Sophisticated nanomachines are used by bacteria for protein secretion. In Gram-negative bacteria, the type 2 secretion system (T2SS) is composed of a pseudopilus assembly platform in the inner ...Sophisticated nanomachines are used by bacteria for protein secretion. In Gram-negative bacteria, the type 2 secretion system (T2SS) is composed of a pseudopilus assembly platform in the inner membrane and a secretin complex in the outer membrane. The engagement of these two megadalton-sized complexes is required in order to secrete toxins, effectors, and hydrolytic enzymes. has at least two T2SSs, with the ancestral nanomachine having a secretin complex composed of XcpQ. Until now, no high-resolution structural information was available to distinguish the features of this -type secretin, which varies greatly in sequence from the well-characterized -type and -type secretins. We have purified the ~1-MDa secretin complex and analyzed it by cryo-electron microscopy. Structural comparisons with the -type secretin complex revealed a striking structural homology despite the differences in their sequence characteristics. At 3.6-Å resolution, the secretin complex was found to have 15-fold symmetry throughout the membrane-embedded region and through most of the domains in the periplasm. However, the N1 domain and N0 domain were not well ordered into this 15-fold symmetry. We suggest a model wherein this disordering of the subunit symmetry for the periplasmic N domains provides a means to engage with the 6-fold symmetry in the inner membrane platform, with a metastable engagement that can be disrupted by substrate proteins binding to the region between XcpP, in the assembly platform, and the XcpQ secretin. How the outer membrane and inner membrane components of the T2SS engage each other and yet can allow for substrate uptake into the secretin chamber has challenged the protein transport field for some time. This vexing question is of significance because the T2SS collects folded protein substrates in the periplasm for transport out of the bacterium and yet must discriminate these few substrate proteins from all the other hundred or so folded proteins in the periplasm. The structural analysis here supports a model wherein substrates must compete against a metastable interaction between XcpP in the assembly platform and the XcpQ secretin, wherein only structurally encoded features in the T2SS substrates compete well enough to disrupt XcpQ-XcpP for entry into the XcpQ chamber, for secretion across the outer membrane.
Validation ReportPDB-ID: 5wln

SummaryFull reportAbout validation report
DateDeposition: Jul 27, 2017 / Header (metadata) release: Oct 18, 2017 / Map release: Oct 25, 2017 / Last update: Jul 18, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.176
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.176
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-5wln
  • Surface level: 0.176
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
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Supplemental images

Downloads & links

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Map

Fileemd_8860.map.gz (map file in CCP4 format, 136049 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
324 pix
1.06 Å/pix.
= 343.44 Å
324 pix
1.06 Å/pix.
= 343.44 Å
324 pix
1.06 Å/pix.
= 343.44 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density
Contour Level:0.176 (by author), 0.176 (movie #1):
Minimum - Maximum-0.8345482 - 1.5014162
Average (Standard dev.)0.0039606583 (0.04376859)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions324324324
Origin0.0.0.
Limit323.323.323.
Spacing324324324
CellA=B=C: 343.43997 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z324324324
origin x/y/z0.0000.0000.000
length x/y/z343.440343.440343.440
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS324324324
D min/max/mean-0.8351.5010.004

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Supplemental data

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Sample components

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Entire Type 2 secretion system outer membrane secretin XcpQ

EntireName: Type 2 secretion system outer membrane secretin XcpQ / Number of components: 2

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Component #1: protein, Type 2 secretion system outer membrane secretin XcpQ

ProteinName: Type 2 secretion system outer membrane secretin XcpQType II secretion system
Recombinant expression: No
MassTheoretical: 1000 kDa
SourceSpecies: Pseudomonas aeruginosa PAO1 (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pET20b / Strain: C43

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Component #2: protein, Type II secretion system protein D

ProteinName: Type II secretion system protein DType II secretion system
Number of Copies: 15 / Recombinant expression: No
MassTheoretical: 66.485656 kDa
SourceSpecies: Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) (bacteria)
Strain: ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1
Source (engineered)Expression System: Escherichia coli (E. coli)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.5 mg/ml / pH: 8
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 4 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 130000. X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Energy filter: GIF
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C15 (15 fold cyclic) / Number of projections: 18005 / Details: MotionCorr 2.1
3D reconstructionCTF correction: CTFFIND4 / Resolution: 3.57 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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Atomic model buiding

Output model

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