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

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Basic information

Entry
Database: EMDB / ID: EMD-8860
TitleCryo-EM structure of the T2SS secretin XcpQ from Pseudomonas aeruginosa
Map dataT2SS secretin XcpQ from Pseudomonas aeruginosa
Sample
  • Complex: Type 2 secretion system outer membrane secretin XcpQ
    • Protein or peptide: Type II secretion system protein D
KeywordsT2SS / Secretin / Type 2 secretion system / Pentadecamer / GspD / XcpQ / MEMBRANE PROTEIN
Function / homology
Function and homology information


protein secretion by the type II secretion system / type II protein secretion system complex / protein secretion / cell outer membrane / identical protein binding
Similarity search - Function
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 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
Similarity search - Domain/homology
Biological speciesPseudomonas 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 / Resolution: 3.57 Å
AuthorsHay ID / Belousoff MJ
Funding support Australia, 2 items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)1092262 Australia
Australian Research Council (ARC)FL130100038). Australia
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.
History
DepositionJul 27, 2017-
Header (metadata) releaseOct 18, 2017-
Map releaseOct 25, 2017-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.176
  • Imaged by UCSF Chimera
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  • 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:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8860.png

Downloads & links

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Map

FileDownload / File: emd_8860.map.gz / Format: CCP4 / Size: 129.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationT2SS secretin XcpQ from Pseudomonas aeruginosa
Projections & slices

Image control

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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.176 / Movie #1: 0.176
Minimum - Maximum-0.8345482 - 1.5014162
Average (Standard dev.)0.0039606583 (±0.04376859)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions324324324
Spacing324324324
CellA=B=C: 343.43997 Å
α=β=γ: 90.0 °

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
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
Components
  • Complex: Type 2 secretion system outer membrane secretin XcpQ
    • Protein or peptide: Type II secretion system protein D

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Supramolecule #1: Type 2 secretion system outer membrane secretin XcpQ

SupramoleculeName: Type 2 secretion system outer membrane secretin XcpQ / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)
Molecular weightTheoretical: 1 MDa

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Macromolecule #1: Type II secretion system protein D

MacromoleculeName: Type II secretion system protein D / type: protein_or_peptide / ID: 1 / Number of copies: 15 / Enantiomer: LEVO
Source (natural)Organism: 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
Molecular weightTheoretical: 66.485656 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: ENSGGNAFVP AGNQQEAHWT INLKDADIRE FIDQISEITG ETFVVDPRVK GQVSVVSKAQ LSLSEVYQLF LSVMSTHGFT VVAQGDQAR IVPNAEAKTE AGGGQSAPDR LETRVIQVQQ SPVSELIPLI RPLVPQYGHL AAVPSANALI ISDRSANIAR I EDVIRQLD ...String:
ENSGGNAFVP AGNQQEAHWT INLKDADIRE FIDQISEITG ETFVVDPRVK GQVSVVSKAQ LSLSEVYQLF LSVMSTHGFT VVAQGDQAR IVPNAEAKTE AGGGQSAPDR LETRVIQVQQ SPVSELIPLI RPLVPQYGHL AAVPSANALI ISDRSANIAR I EDVIRQLD QKGSHDYSVI NLRYGWVMDA AEVLNNAMSR GQAKGAAGAQ VIADARTNRL IILGPPQARA KLVQLAQSLD TP TARSANT RVIRLRHNDA KTLAETLGQI SEGMKNNGGQ GGEQTGGGRP SNILIRADES TNALVLLADP DTVNALEDIV RQL DVPRAQ VLVEAAIVEI SGDIQDAVGV QWAINKGGMG GTKTNFANTG LSIGTLLQSL ESNKAPESIP DGAIVGIGSS SFGA LVTAL SANTKSNLLS TPSLLTLDNQ KAEILVGQNV PFQTGSYTTN SEGSSNPFTT VERKDIGVSL KVTPHINDGA ALRLE IEQE ISALLPNAQQ RNNTDLITSK RSIKSTILAE NGQVIVIGGL IQDDVSQAES KVPLLGDIPL LGRLFRSTKD THTKRN LMV FLRPTVVRDS AGLAALSGKK YSDIRVIDGT RGPEGRPSIL PTNANQLFDG QAVDLRELMT E

UniProtKB: Secretin XcpQ

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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

Concentration0.5 mg/mL
BufferpH: 8 / Component:
ConcentrationNameFormula
20.0 mMHEPES
300.0 mMNaCl
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK II

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 2.5 µm / Calibrated defocus min: 0.6 µm / Calibrated magnification: 130000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsMotionCorr 2.1
Particle selectionNumber selected: 18005
Startup modelType of model: INSILICO MODEL / In silico model: Cryosparc Ab-initio
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C15 (15 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.57 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 0.4.1) / Number images used: 18005
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 0.4.1)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 0.4.1)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 0.4.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: FLEXIBLE FIT
Output model

PDB-5wln:
Cryo-EM structure of the T2SS secretin XcpQ from Pseudomonas aeruginosa

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