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- PDB-8axn: Inner membrane ring and secretin N0 N1 domains of the type 3 secr... -

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Basic information

Entry
Database: PDB / ID: 8axn
TitleInner membrane ring and secretin N0 N1 domains of the type 3 secretion system of Shigella flexneri
Components
  • Lipoprotein MxiJ
  • Outer membrane protein MxiD
  • Protein MxiG
KeywordsPROTEIN TRANSPORT / type 3 secretion / inner membrane ring / secretin / shigella / infection
Function / homology
Function and homology information


type III protein secretion system complex / protein secretion by the type III secretion system / protein secretion / cell outer membrane / plasma membrane
Similarity search - Function
Type III secretion system lipoprotein HrcJ/YscJ / Type III secretion system, PrgH/EprH-like / Type III secretion system protein PrgH-EprH (PrgH) / : / SPI-1 type 3 secretion system secretin, N0 domain / Type III secretion system outer membrane pore YscC/HrcC / 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 ...Type III secretion system lipoprotein HrcJ/YscJ / Type III secretion system, PrgH/EprH-like / Type III secretion system protein PrgH-EprH (PrgH) / : / SPI-1 type 3 secretion system secretin, N0 domain / Type III secretion system outer membrane pore YscC/HrcC / 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 / Lipoprotein YscJ/Flagellar M-ring protein / Secretory protein of YscJ/FliF family / Flagellar M-ring , N-terminal / AMP-binding enzyme, C-terminal domain superfamily / Prokaryotic membrane lipoprotein lipid attachment site profile.
Similarity search - Domain/homology
Protein MxiG / Type 3 secretion system secretin / Lipoprotein MxiJ
Similarity search - Component
Biological speciesShigella flexneri (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.34 Å
AuthorsLunelli, M.
Funding support Germany, 1items
OrganizationGrant numberCountry
Helmholtz Association Germany
CitationJournal: Protein Sci / Year: 2023
Title: Integrative structural analysis of the type III secretion system needle complex from Shigella flexneri.
Authors: Lara Flacht / Michele Lunelli / Karol Kaszuba / Zhuo Angel Chen / Francis J O' Reilly / Juri Rappsilber / Jan Kosinski / Michael Kolbe /
Abstract: The type III secretion system (T3SS) is a large, transmembrane protein machinery used by various pathogenic gram-negative bacteria to transport virulence factors into the host cell during infection. ...The type III secretion system (T3SS) is a large, transmembrane protein machinery used by various pathogenic gram-negative bacteria to transport virulence factors into the host cell during infection. Understanding the structure of T3SSs is crucial for future developments of therapeutics that could target this system. However, much of the knowledge about the structure of T3SS is available only for Salmonella, and it is unclear how this large assembly is conserved across species. Here, we combined cryo-electron microscopy, cross-linking mass spectrometry, and integrative modeling to determine the structure of the T3SS needle complex from Shigella flexneri. We show that the Shigella T3SS exhibits unique features distinguishing it from other structurally characterized T3SSs. The secretin pore complex adopts a new fold of its C-terminal S domain and the pilotin MxiM[SctG] locates around the outer surface of the pore. The export apparatus structure exhibits a conserved pseudohelical arrangement but includes the N-terminal domain of the SpaS[SctU] subunit, which was not present in any of the previously published virulence-related T3SS structures. Similar to other T3SSs, however, the apparatus is anchored within the needle complex by a network of flexible linkers that either adjust conformation to connect to equivalent patches on the secretin oligomer or bind distinct surface patches at the same height of the export apparatus. The conserved and unique features delineated by our analysis highlight the necessity to analyze T3SS in a species-specific manner, in order to fully understand the underlying molecular mechanisms of these systems. The structure of the type III secretion system from Shigella flexneri delineates conserved and unique features, which could be used for the development of broad-range therapeutics.
History
DepositionAug 31, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 1, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 12, 2023Group: Database references / Category: citation / citation_author / Item: _citation.journal_volume / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: Protein MxiG
B: Protein MxiG
A: Protein MxiG
a: Lipoprotein MxiJ
0: Outer membrane protein MxiD
1: Outer membrane protein MxiD
d: Lipoprotein MxiJ
c: Lipoprotein MxiJ
S: Protein MxiG
P: Protein MxiG
M: Protein MxiG
J: Protein MxiG
G: Protein MxiG
D: Protein MxiG
U: Protein MxiG
T: Protein MxiG
Q: Protein MxiG
N: Protein MxiG
K: Protein MxiG
H: Protein MxiG
E: Protein MxiG
V: Protein MxiG
R: Protein MxiG
O: Protein MxiG
L: Protein MxiG
I: Protein MxiG
F: Protein MxiG
W: Protein MxiG
X: Protein MxiG
b: Lipoprotein MxiJ
e: Lipoprotein MxiJ
h: Lipoprotein MxiJ
k: Lipoprotein MxiJ
n: Lipoprotein MxiJ
q: Lipoprotein MxiJ
t: Lipoprotein MxiJ
x: Lipoprotein MxiJ
g: Lipoprotein MxiJ
j: Lipoprotein MxiJ
m: Lipoprotein MxiJ
p: Lipoprotein MxiJ
s: Lipoprotein MxiJ
w: Lipoprotein MxiJ
v: Lipoprotein MxiJ
u: Lipoprotein MxiJ
f: Lipoprotein MxiJ
i: Lipoprotein MxiJ
l: Lipoprotein MxiJ
o: Lipoprotein MxiJ
r: Lipoprotein MxiJ
a0: Outer membrane protein MxiD
Y: Outer membrane protein MxiD
Z: Outer membrane protein MxiD
2: Outer membrane protein MxiD
4: Outer membrane protein MxiD
6: Outer membrane protein MxiD
8: Outer membrane protein MxiD
b0: Outer membrane protein MxiD
y: Outer membrane protein MxiD
z: Outer membrane protein MxiD
3: Outer membrane protein MxiD
5: Outer membrane protein MxiD
7: Outer membrane protein MxiD
9: Outer membrane protein MxiD


Theoretical massNumber of molelcules
Total (without water)2,705,98864
Polymers2,705,98864
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein ...
Protein MxiG


Mass: 43053.844 Da / Num. of mol.: 24 / Source method: isolated from a natural source / Source: (natural) Shigella flexneri (bacteria) / References: UniProt: P0A221
#2: Protein ...
Lipoprotein MxiJ


Mass: 27542.055 Da / Num. of mol.: 24 / Source method: isolated from a natural source / Source: (natural) Shigella flexneri (bacteria) / References: UniProt: Q06081
#3: Protein
Outer membrane protein MxiD


Mass: 63230.414 Da / Num. of mol.: 16 / Source method: isolated from a natural source / Source: (natural) Shigella flexneri (bacteria) / References: UniProt: Q04641

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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

Component
IDNameTypeEntity IDParent-IDSourceDetails
1Needle complex of the type 3 secretion systemCOMPLEXall0NATURAL
2Inner membrane ringCOMPLEX#1-#21NATURAL
3ConnectorCOMPLEX#31NATURALRing formed by the domains N0 and N1 of the secretin MxiD
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Shigella flexneri (bacteria)623
32Shigella flexneri (bacteria)623
43Shigella flexneri (bacteria)623
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 101179 X / Nominal defocus max: 4000 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 25 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5238
Image scansWidth: 4096 / Height: 4096 / Movie frames/image: 7 / Used frames/image: 1-6

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4.1.13CTF correction
7UCSF Chimera1.15model fitting
9PHENIX1.18.2_3874model refinement
10RELION3initial Euler assignment
11RELION3final Euler assignment
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C8 (8 fold cyclic)
3D reconstructionResolution: 3.34 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 90547 / Details: Focused reconstruction / Symmetry type: POINT
Atomic model buildingB value: 67 / Protocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6RWK

6rwk

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00396240
ELECTRON MICROSCOPYf_angle_d0.508129992
ELECTRON MICROSCOPYf_dihedral_angle_d18.47536512
ELECTRON MICROSCOPYf_chiral_restr0.04314800
ELECTRON MICROSCOPYf_plane_restr0.00316616

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