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- PDB-8axl: Outer membrane secretin pore of the type 3 secretion system of Sh... -

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Basic information

Entry
Database: PDB / ID: 8axl
TitleOuter membrane secretin pore of the type 3 secretion system of Shigella flexneri
ComponentsOuter membrane protein MxiD
KeywordsPROTEIN TRANSPORT / type 3 secretion / secretin / outer membrane ring / shigella / infection
Function / homology
Function and homology information


type III protein secretion system complex / protein secretion by the type III secretion system / cell outer membrane
Similarity search - Function
: / 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 / 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
Type 3 secretion system secretin
Similarity search - Component
Biological speciesShigella flexneri (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.42 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
Revision 1.2Jul 24, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update
Revision 1.3Nov 13, 2024Group: Data collection / Structure summary
Category: em_admin / em_entity_assembly_molwt / pdbx_entry_details
Item: _em_admin.last_update / _em_entity_assembly_molwt.id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Outer membrane protein MxiD
O: Outer membrane protein MxiD
B: Outer membrane protein MxiD
C: Outer membrane protein MxiD
D: Outer membrane protein MxiD
E: Outer membrane protein MxiD
F: Outer membrane protein MxiD
G: Outer membrane protein MxiD
H: Outer membrane protein MxiD
I: Outer membrane protein MxiD
J: Outer membrane protein MxiD
K: Outer membrane protein MxiD
L: Outer membrane protein MxiD
M: Outer membrane protein MxiD
N: Outer membrane protein MxiD


Theoretical massNumber of molelcules
Total (without water)948,45615
Polymers948,45615
Non-polymers00
Water00
1


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

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Components

#1: Protein
Outer membrane protein MxiD


Mass: 63230.414 Da / Num. of mol.: 15 / Source method: isolated from a natural source / Source: (natural) Shigella flexneri (bacteria) / References: UniProt: Q04641
Has protein modificationN

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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
2Outer membrane secretin pore of the type 3 secretion systemCOMPLEXall1NATURALPore formed by the oligomerization of the domains N3, Secretin and S of MxiD
Molecular weight
IDEntity assembly-IDExperimental value
11NO
22
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Shigella flexneri (bacteria)623
22Shigella 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 FIELD / 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
9RELION3initial Euler assignment
10RELION3final Euler assignment
12RELION33D reconstruction
13PHENIX1.18.2_3874model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C15 (15 fold cyclic)
3D reconstructionResolution: 3.42 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 69589
Details: Focused reconstruction obtained after partial signal subtraction.
Symmetry type: POINT
Atomic model buildingB value: 72.8 / Protocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00638160
ELECTRON MICROSCOPYf_angle_d0.6351615
ELECTRON MICROSCOPYf_dihedral_angle_d15.53614265
ELECTRON MICROSCOPYf_chiral_restr0.0476150
ELECTRON MICROSCOPYf_plane_restr0.0056600

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