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- PDB-6f2d: A FliPQR complex forms the core of the Salmonella type III secret... -

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Basic information

Entry
Database: PDB / ID: 6f2d
TitleA FliPQR complex forms the core of the Salmonella type III secretion system export apparatus.
Components
  • Flagellar biosynthetic protein FliP
  • Flagellar biosynthetic protein FliQ
  • Flagellar biosynthetic protein FliR
KeywordsPROTEIN TRANSPORT / T3SS / Flagella / Cryo-EM / Export / Membrane protein
Function / homology
Function and homology information


bacterial-type flagellum organization / bacterial-type flagellum basal body / bacterial-type flagellum assembly / protein secretion / protein targeting / membrane => GO:0016020 / plasma membrane
Similarity search - Function
Flagellar biosynthesis protein FliQ / Flagellar biosynthesis protein FliR / Flagellar transport protein FliP / Type III secretion system inner membrane R protein / Bacterial export protein family 3 / Bacterial export proteins, family 1 / Bacterial export proteins, family 3 / Flagella transport protein fliP family signature 1. / Type III secretion system inner membrane P protein / FliP family / Flagella transport protein fliP family signature 2.
Similarity search - Domain/homology
Flagellar biosynthetic protein FliP / Flagellar biosynthetic protein FliQ / Flagellar biosynthetic protein FliQ / Flagellar biosynthetic protein FliP / Flagellar biosynthetic protein FliR
Similarity search - Component
Biological speciesSalmonella enterica subsp. enterica (bacteria)
Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsJohnson, S. / Kuhlen, L. / Abrusci, P. / Lea, S.M.
Funding support United Kingdom, 4items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)M011984 United Kingdom
Wellcome Trust100298 United Kingdom
Wellcome Trust201536 United Kingdom
Wolfson FoundationWL160052 United 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 / ...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.
History
DepositionNov 24, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 4, 2018Provider: repository / Type: Initial release
Revision 1.1Jul 18, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name
Revision 1.2Jul 25, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Dec 11, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Structure visualization

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Assembly

Deposited unit
A: Flagellar biosynthetic protein FliP
B: Flagellar biosynthetic protein FliP
C: Flagellar biosynthetic protein FliP
D: Flagellar biosynthetic protein FliP
E: Flagellar biosynthetic protein FliP
F: Flagellar biosynthetic protein FliR
G: Flagellar biosynthetic protein FliQ
H: Flagellar biosynthetic protein FliQ
I: Flagellar biosynthetic protein FliQ
J: Flagellar biosynthetic protein FliQ


Theoretical massNumber of molelcules
Total (without water)205,34110
Polymers205,34110
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: light scattering
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area38740 Å2
ΔGint-497 kcal/mol
Surface area76400 Å2
MethodPISA

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Components

#1: Protein
Flagellar biosynthetic protein FliP


Mass: 26769.021 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Salmonella enterica subsp. enterica (bacteria)
Gene: fliP, LTSERUB_0568 / Production host: Escherichia coli (E. coli) / Strain (production host): Mt56 / References: UniProt: G5QE81, UniProt: P54700*PLUS
#2: Protein Flagellar biosynthetic protein FliR


Mass: 33069.180 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Residues 265 onwards constitute the Twin-Strep tag used for purification
Source: (gene. exp.) Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Strain: LT2 / SGSC1412 / ATCC 700720 / Gene: fliR, flaP, STM1981 / Production host: Escherichia coli (E. coli) / Strain (production host): Mt56 / References: UniProt: P54702
#3: Protein
Flagellar biosynthetic protein FliQ


Mass: 9606.758 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Gene: fliQ, flaQ, STY2188, t0897 / Production host: Escherichia coli (E. coli) / Strain (production host): Mt56 / References: UniProt: P0A1L6, UniProt: P0A1L5*PLUS

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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: Complex of the flagellar type III secretion system export apparatus components FliP, FliQ and FliRType three secretion system
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 195 kDa/nm / Experimental value: YES
Source (natural)Organism: Salmonella enterica (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: Mt56
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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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 / Nominal defocus max: 4000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recording
IDImaging-IDElectron dose (e/Å2)Detector modeFilm or detector modelNum. of real images
1147COUNTINGGATAN K2 SUMMIT (4k x 4k)401
2150COUNTINGFEI FALCON III (4k x 4k)1687

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Processing

SoftwareName: PHENIX / Version: 1.10_2155: / Classification: refinement
EM software
IDNameVersionCategory
1Gautomatchparticle selection
4CTFFIND4.1.8CTF correction
10RELION2initial Euler assignment
11RELION2final Euler assignment
13RELION23D reconstruction
Image processingDetails: Images from K2 were also added.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 97718 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0112820
ELECTRON MICROSCOPYf_angle_d1.17517476
ELECTRON MICROSCOPYf_dihedral_angle_d6.61510451
ELECTRON MICROSCOPYf_chiral_restr0.0542189
ELECTRON MICROSCOPYf_plane_restr0.0082130

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