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- PDB-6s3l: Structure of the core of the flagellar export apparatus from Vibr... -

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Basic information

Entry
Database: PDB / ID: 6s3l
TitleStructure of the core of the flagellar export apparatus from Vibrio mimicus, the FliPQR-FlhB complex.
Components
  • Flagellar biosynthetic protein FlhB
  • 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 / plasma membrane
Similarity search - Function
Flagellar biosynthetic protein FlhB / Type III secretion system substrate exporter / Type III secretion system substrate exporter, C-terminal / FlhB HrpN YscU SpaS Family / Flagellar biosynthesis protein FliQ / Flagellar transport protein FliP / Flagellar biosynthesis protein FliR / Bacterial export protein family 3 / Bacterial export proteins, family 3 / Flagella transport protein fliP family signature 1. ...Flagellar biosynthetic protein FlhB / Type III secretion system substrate exporter / Type III secretion system substrate exporter, C-terminal / FlhB HrpN YscU SpaS Family / Flagellar biosynthesis protein FliQ / Flagellar transport protein FliP / Flagellar biosynthesis protein FliR / Bacterial export protein family 3 / 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. / Type III secretion system inner membrane R protein / Bacterial export proteins, family 1
Similarity search - Domain/homology
Flagellar biosynthetic protein FliQ / Flagellar biosynthetic protein FlhB / Flagellar biosynthetic protein FliR / Flagellar biosynthetic protein FliP
Similarity search - Component
Biological speciesVibrio mimicus CAIM 602 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 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
Revision 1.1May 22, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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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 FlhB


Theoretical massNumber of molelcules
Total (without water)278,52111
Polymers278,52111
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: light scattering
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area53560 Å2
ΔGint-635 kcal/mol
Surface area62050 Å2
MethodPISA

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Components

#1: Protein
Flagellar biosynthetic protein FliP


Mass: 32431.307 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio mimicus CAIM 602 (bacteria) / Gene: fliP, AL544_009685 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: A0A2J9UXT5
#2: Protein Flagellar biosynthetic protein FliR


Mass: 28764.324 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio mimicus CAIM 602 (bacteria) / Gene: AL544_009675 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A1D8S9I5
#3: Protein
Flagellar biosynthetic protein FliQ


Mass: 10333.578 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio mimicus CAIM 602 (bacteria) / Gene: fliQ, AL544_009680 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A1D8S9F5
#4: Protein Flagellar biosynthetic protein FlhB


Mass: 46265.730 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vibrio mimicus CAIM 602 (bacteria) / Gene: flhB, AL544_009670 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A1D8S9F8

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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-FlhB / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.26 MDa / Experimental value: YES
Source (natural)Organism: Vibrio mimicus CAIM 602 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21 (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMTrisC4H11NO31
2150 mMsodium chlorideNaCl1
31 mMEDTAC10H16N2O81
40.01 %LMNGC47H88O221
SpecimenConc.: 2.5 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

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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 FIELD / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
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
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1386230
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 162408 / Symmetry type: POINT
RefinementHighest resolution: 3.2 Å
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0114138
ELECTRON MICROSCOPYf_angle_d0.868719178
ELECTRON MICROSCOPYf_chiral_restr0.05072359
ELECTRON MICROSCOPYf_plane_restr0.00662329
ELECTRON MICROSCOPYf_dihedral_angle_d11.63058491

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