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- PDB-6s3r: Structure of the FliPQR complex from the flagellar type 3 secreti... -

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Basic information

Entry
Database: PDB / ID: 6s3r
TitleStructure of the FliPQR complex from the flagellar type 3 secretion system of Pseudomonas savastanoi.
Components
  • Flagellar biosynthetic protein FliP
  • Flagellar biosynthetic protein FliQ
  • Flagellar biosynthetic protein FliR
KeywordsPROTEIN TRANSPORT / flagella / T3SS / export apparatus / export gate
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 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 FliR / Flagellar biosynthetic protein FliP / Flagellar biosynthetic protein FliQ
Similarity search - Component
Biological speciesPseudomonas savastanoi pv. phaseolicola (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsKuhlen, L. / Johnson, S. / Deme, J.C. / Lea, S.M.
Funding support United Kingdom, 5items
OrganizationGrant numberCountry
Wellcome Trust100298 United Kingdom
Wellcome Trust109136 United Kingdom
Wellcome Trust201536 United Kingdom
Medical Research Council (United Kingdom)M011984 United Kingdom
Wolfson FoundationWL160052 United Kingdom
CitationJournal: Nat Commun / Year: 2020
Title: The substrate specificity switch FlhB assembles onto the export gate to regulate type three secretion.
Authors: Lucas Kuhlen / Steven Johnson / Andreas Zeitler / Sandra Bäurle / Justin C Deme / Joseph J E Caesar / Rebecca Debo / Joseph Fisher / Samuel Wagner / Susan M Lea /
Abstract: Protein secretion through type-three secretion systems (T3SS) is critical for motility and virulence of many bacteria. Proteins are transported through an export gate containing three proteins ...Protein secretion through type-three secretion systems (T3SS) is critical for motility and virulence of many bacteria. Proteins are transported through an export gate containing three proteins (FliPQR in flagella, SctRST in virulence systems). A fourth essential T3SS protein (FlhB/SctU) functions to "switch" secretion substrate specificity once the growing hook/needle reach their determined length. Here, we present the cryo-electron microscopy structure of an export gate containing the switch protein from a Vibrio flagellar system at 3.2 Å resolution. The structure reveals that FlhB/SctU extends the helical export gate with its four predicted transmembrane helices wrapped around FliPQR/SctRST. The unusual topology of the FlhB/SctU helices creates a loop wrapped around the bottom of the closed export gate. Structure-informed mutagenesis suggests that this loop is critical in gating secretion and we propose that a series of conformational changes in the T3SS trigger opening of the gate through interactions between FlhB/SctU and FliPQR/SctRST.
History
DepositionJun 25, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 25, 2020Provider: repository / Type: Initial release

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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
K: Flagellar biosynthetic protein FliQ


Theoretical massNumber of molelcules
Total (without water)217,97711
Polymers217,97711
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by software
  • Evidence: mass spectrometry, native mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area47470 Å2
ΔGint-589 kcal/mol
Surface area61990 Å2
MethodPISA

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Components

#1: Protein
Flagellar biosynthetic protein FliP


Mass: 27199.805 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas savastanoi pv. phaseolicola (strain 1448A / Race 6) (bacteria)
Strain: 1448A / Race 6 / Gene: fliP, PSPPH_3370 / Production host: Escherichia coli (E. coli) / References: UniProt: Q48GF5
#2: Protein Flagellar biosynthetic protein FliR


Mass: 32352.314 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas savastanoi pv. phaseolicola (bacteria)
Gene: ALQ02_02028 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0P9WRJ4
#3: Protein
Flagellar biosynthetic protein FliQ


Mass: 9925.104 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas savastanoi pv. phaseolicola (strain 1448A / Race 6) (bacteria)
Strain: 1448A / Race 6 / Gene: fliQ, PSPPH_3369 / Production host: Escherichia coli (E. coli) / References: UniProt: Q48GF6

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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: FliPQR complex from the flagellar type 3 secretion system of Pseudomonas savastanoi
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.21 MDa / Experimental value: YES
Source (natural)Organism: Pseudomonas savastanoi pv. phaseolicola 1448A (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMTrisC4H11NO31
2150 mMsodium chlorideNaClSodium chloride1
31 mMEDTAEthylenediaminetetraacetic acidC10H16N2O81
40.01 %LMNGC47H88O221
SpecimenConc.: 4.1 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 R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K / Details: 10 seconds wait time before blotting

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 165000 X / Nominal defocus max: 4000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 48 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

Software
NameVersionClassificationNB
PHENIX1.15.2_3472refinement
PHENIX1.15.2_3472refinement
EM software
IDNameCategory
1SIMPLEparticle selection
2EPUimage acquisition
4CTFFINDCTF correction
11RELIONclassification
12RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 503177
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 97987 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
RefinementStereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.005713117
ELECTRON MICROSCOPYf_angle_d0.840517871
ELECTRON MICROSCOPYf_chiral_restr0.04622288
ELECTRON MICROSCOPYf_plane_restr0.00672149
ELECTRON MICROSCOPYf_dihedral_angle_d10.39237900

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