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- EMDB-10095: Structure of the FliPQR complex from the flagellar type 3 secreti... -

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Basic information

Entry
Database: EMDB / ID: EMD-10095
TitleStructure of the FliPQR complex from the flagellar type 3 secretion system of Pseudomonas savastanoi.
Map dataStructure of the FliPQR complex from the flagellar type 3 secretion system of Pseudomonas savastanoi.
Sample
  • Complex: FliPQR complex from the flagellar type 3 secretion system of Pseudomonas savastanoi
    • Protein or peptide: Flagellar biosynthetic protein FliP
    • Protein or peptide: Flagellar biosynthetic protein FliR
    • Protein or peptide: Flagellar biosynthetic protein FliQ
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 1448A (bacteria) / Pseudomonas savastanoi pv. phaseolicola (strain 1448A / Race 6) (bacteria) / Pseudomonas savastanoi pv. phaseolicola (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsKuhlen L / Johnson S / Deme JC / Lea SM
Funding support United Kingdom, 5 items
OrganizationGrant numberCountry
Wellcome Trust100298 United Kingdom
Wellcome Trust201536 United Kingdom
Wellcome Trust109136 United Kingdom
Wolfson FoundationWL160052 United Kingdom
Medical Research Council (United Kingdom)M011984 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, 2019-
Header (metadata) releaseJul 10, 2019-
Map releaseMar 25, 2020-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0166
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0166
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6s3r
  • Surface level: 0.0166
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10095.png

Downloads & links

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Map

FileDownload / File: emd_10095.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of the FliPQR complex from the flagellar type 3 secretion system of Pseudomonas savastanoi.
Voxel sizeX=Y=Z: 0.822 Å
Density
Contour LevelBy AUTHOR: 0.0166 / Movie #1: 0.0166
Minimum - Maximum-0.052240472 - 0.081255555
Average (Standard dev.)0.00000248061 (±0.0031633396)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 236.73601 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8220.8220.822
M x/y/z288288288
origin x/y/z0.0000.0000.000
length x/y/z236.736236.736236.736
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS288288288
D min/max/mean-0.0520.0810.000

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Supplemental data

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Mask #1

Fileemd_10095_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: refinement map

Fileemd_10095_additional.map
Annotationrefinement map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map

Fileemd_10095_half_map_1.map
Annotationhalf map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map

Fileemd_10095_half_map_2.map
Annotationhalf map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : FliPQR complex from the flagellar type 3 secretion system of Pseu...

EntireName: FliPQR complex from the flagellar type 3 secretion system of Pseudomonas savastanoi
Components
  • Complex: FliPQR complex from the flagellar type 3 secretion system of Pseudomonas savastanoi
    • Protein or peptide: Flagellar biosynthetic protein FliP
    • Protein or peptide: Flagellar biosynthetic protein FliR
    • Protein or peptide: Flagellar biosynthetic protein FliQ

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Supramolecule #1: FliPQR complex from the flagellar type 3 secretion system of Pseu...

SupramoleculeName: FliPQR complex from the flagellar type 3 secretion system of Pseudomonas savastanoi
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Pseudomonas savastanoi pv. phaseolicola 1448A (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightExperimental: 210 KDa

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Macromolecule #1: Flagellar biosynthetic protein FliP

MacromoleculeName: Flagellar biosynthetic protein FliP / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas savastanoi pv. phaseolicola (strain 1448A / Race 6) (bacteria)
Strain: 1448A / Race 6
Molecular weightTheoretical: 27.199805 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGALRFVILL LLVMVTPAVL AADPLSIPAI TLSNGADGQQ EYSVSLQILL IMTALSFIPA FVMLMTSFTR IIIVFSILRQ ALGLQQTPS NQILTGMALF LTMFIMAPVF DRVNQDALQP YLAEKLSAQD AVAKAQVPIK DFMLAQTRTS DLELFMRLSK R TDIPTPDA ...String:
MGALRFVILL LLVMVTPAVL AADPLSIPAI TLSNGADGQQ EYSVSLQILL IMTALSFIPA FVMLMTSFTR IIIVFSILRQ ALGLQQTPS NQILTGMALF LTMFIMAPVF DRVNQDALQP YLAEKLSAQD AVAKAQVPIK DFMLAQTRTS DLELFMRLSK R TDIPTPDA APLTILVPAF VISELKTAFQ IGFMIFIPFL IIDLVVASVL MAMGMMMLSP LIISLPFKIM LFVLVDGWAL IV GTLAGSF GGV

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Macromolecule #2: Flagellar biosynthetic protein FliR

MacromoleculeName: Flagellar biosynthetic protein FliR / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas savastanoi pv. phaseolicola (bacteria)
Molecular weightTheoretical: 32.352314 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MQPMLALTDI QISTWVASFM LPMFRIVALL MTMPVIGTTL VPRRVRLYLA FAITVVVAPA LPAMPPVQAL DLSGLLLIGE QIIIGAGMG LSLQMFFHIF VIAGQIISTQ MGMGFASMVD PTNGVSSAVI GQFFTMLVTL LFLFMNGHLV VLEVLVESFT T MPVGGGLL ...String:
MQPMLALTDI QISTWVASFM LPMFRIVALL MTMPVIGTTL VPRRVRLYLA FAITVVVAPA LPAMPPVQAL DLSGLLLIGE QIIIGAGMG LSLQMFFHIF VIAGQIISTQ MGMGFASMVD PTNGVSSAVI GQFFTMLVTL LFLFMNGHLV VLEVLVESFT T MPVGGGLL VNNFWELANG LGWALSSGLR LVLPAITALL IINIAFGVMT RAAPQLNIFS IGFPLTLVLG MVILWMSMGD IL NQYQPIA SQALQSLRDM VRARENLYFQ GQFGSWSHPQ FEKGGGSGGG SGGGSWSHPQ FEK

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Macromolecule #3: Flagellar biosynthetic protein FliQ

MacromoleculeName: Flagellar biosynthetic protein FliQ / type: protein_or_peptide / ID: 3 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas savastanoi pv. phaseolicola (strain 1448A / Race 6) (bacteria)
Strain: 1448A / Race 6
Molecular weightTheoretical: 9.925104 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MTPEVAVDLF REALWLTTVL VAILVVPSLL CGLLVAMFQA ATQINEQTLS FLPRLLVMLV TLIVIGPWLL KIFMEYMLSL YTSIPTLIG

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration4.1 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
100.0 mMC4H11NO3Tris
150.0 mMNaClSodium chloridesodium chloride
1.0 mMC10H16N2O8EDTAEthylenediaminetetraacetic acid
0.01 %C47H88O22LMNG
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV / Details: 10 seconds wait time before blotting.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 165000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 48.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 503177
CTF correctionSoftware - Name: CTFFIND
Startup modelType of model: EMDB MAP
EMDB ID:

4173

Initial angle assignmentType: PROJECTION MATCHING
Final 3D classificationNumber classes: 5 / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 97987
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-6s3r:
Structure of the FliPQR complex from the flagellar type 3 secretion system of Pseudomonas savastanoi.

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