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- PDB-6r6b: Structure of the core Shigella flexneri type III secretion system... -

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Entry
Database: PDB / ID: 6r6b
TitleStructure of the core Shigella flexneri type III secretion system export gate complex SctRST (Spa24/Spa9/Spa29).
Components
  • Surface presentation of antigens protein SpaP
  • Surface presentation of antigens protein SpaQ
  • Surface presentation of antigens protein SpaR
KeywordsPROTEIN TRANSPORT / Type III secretion / T3SS / Flagella / Cryo-EM / Membrane protein / Protein transport
Function / homology
Function and homology information


protein secretion / protein targeting / pathogenesis / integral component of membrane / plasma membrane
Type III secretion protein SpaR/YscT / Flagella transport protein fliP family signature 2. / Type III secretion system inner membrane R protein / Bacterial export protein family 3 / Yop virulence translocation protein R / Type III secretion system inner membrane P protein / Type III secretion protein HrpO / FliP family / Bacterial export proteins, family 1 / Bacterial export proteins, family 3 / Flagella transport protein fliP family signature 1.
Surface presentation of antigens protein SpaP / Surface presentation of antigens protein SpaQ / Surface presentation of antigens protein SpaR
Biological speciesShigella flexneri (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsJohnson, S. / Kuhlen, L. / Deme, J.C. / Abrusci, P. / Lea, S.M.
Funding supportUnited Kingdom , 5件
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)M011984United Kingdom
Wellcome Trust201536United Kingdom
Wellcome Trust109136United Kingdom
Wellcome Trust201536United Kingdom
Wolfson FoundationWL160052United Kingdom
CitationJournal: Nat. Struct. Mol. Biol. / Year: 2018
Title: Structure of the core of the type III secretion system export apparatus.
Authors: Lucas Kuhlen / Patrizia Abrusci / Steven Johnson / Joseph Gault / Justin Deme / Joseph Caesar / Tobias Dietsche / Mehari Tesfazgi Mebrhatu / Tariq Ganief / Boris Macek / Samuel Wagner / Carol V Robinson / Susan M Lea /
Abstract: Export of proteins through type III secretion systems is critical for motility and virulence of many major bacterial pathogens. Three putative integral membrane proteins (FliP, FliQ, FliR) are ...Export of proteins through type III secretion systems is critical for motility and virulence of many major bacterial pathogens. Three putative integral membrane proteins (FliP, FliQ, FliR) are suggested to form the core of an export gate in the inner membrane, but their structure, assembly and location within the final nanomachine remain unclear. Here, we present the cryoelectron microscopy structure of the Salmonella Typhimurium FliP-FliQ-FliR complex at 4.2 Å. None of the subunits adopt canonical integral membrane protein topologies, and common helix-turn-helix structural elements allow them to form a helical assembly with 5:4:1 stoichiometry. Fitting of the structure into reconstructions of intact secretion systems, combined with cross-linking, localize the export gate as a core component of the periplasmic portion of the machinery. This study thereby identifies the export gate as a key element of the secretion channel and implies that it primes the helical architecture of the components assembling downstream.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Mar 26, 2019 / Release: May 29, 2019
RevisionDateData content typeProviderType
1.0May 29, 2019Structure modelrepositoryInitial release

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Assembly

Deposited unit
A: Surface presentation of antigens protein SpaP
B: Surface presentation of antigens protein SpaP
C: Surface presentation of antigens protein SpaP
D: Surface presentation of antigens protein SpaP
E: Surface presentation of antigens protein SpaP
F: Surface presentation of antigens protein SpaR
I: Surface presentation of antigens protein SpaQ
G: Surface presentation of antigens protein SpaQ
H: Surface presentation of antigens protein SpaQ
J: Surface presentation of antigens protein SpaQ


Theoretical massNumber of molelcules
Total (without water)191,45510
Polymers191,45510
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area43470 Å2
ΔGint-493 kcal/mol
Surface area55080 Å2
MethodPISA

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Components

#1: Protein/peptide
Surface presentation of antigens protein SpaP / Spa24 protein


Mass: 24215.562 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Shigella flexneri (bacteria) / Gene: spaP, spa24, CP0153 / Production host: Escherichia coli (E. coli) / Variant (production host): MT56 / References: UniProt: P0A1L3
#2: Protein/peptide Surface presentation of antigens protein SpaR / Spa29 protein


Mass: 32644.086 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Shigella flexneri (bacteria) / Gene: spaR, spa29, CP0155 / Production host: Escherichia coli (E. coli) / Variant (production host): MT56 / References: UniProt: P0A1M6
#3: Protein/peptide
Surface presentation of antigens protein SpaQ / Protein spa9


Mass: 9433.338 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Shigella flexneri (bacteria) / Gene: spaQ, spa9, CP0154 / Production host: Escherichia coli (E. coli) / Variant (production host): MT56 / References: UniProt: P0A1M4

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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

ComponentName: Shigella flexneri type III secretion export gate (Spa24Spa9Spa29)Type three secretion system
Type: COMPLEX / Entity ID: 1,2,3 / Source: RECOMBINANT
Molecular weightValue: 0.191 MDa / Experimental value: YES
Source (natural)Organism: Shigella flexneri (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: MT56
Buffer solutionpH: 8
Buffer component

Buffer-ID: 1

IDConc.Name
1100 mMTris
2150 mMsodium chloride
31 mMEDTAEthylenediaminetetraacetic acid
SpecimenConc.: 8.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
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: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 48 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 3741
Image scansMovie frames/image: 20

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Processing

EM software
IDNameVersionCategory
4CTFFIND4.1.8CTF correction
7Buccaneermodel fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
12RELION33D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 775073
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 212561 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL

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