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- EMDB-10096: Structure of the FliPQR complex from the flagellar type 3 secreti... -

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Basic information

Entry
Database: EMDB / ID: EMD-10096
TitleStructure of the FliPQR complex from the flagellar type 3 secretion system of Vibrio mimicus.
Map dataStructure of the FliPQR complex from the flagellar type 3 secretion system of Vibrio mimicus.
Sample
  • Complex: FliPQR
    • 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 / 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 FliQ / Flagellar biosynthetic protein FliR / Flagellar biosynthetic protein FliP
Similarity search - Component
Biological speciesVibrio mimicus CAIM 602 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 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) releaseMar 25, 2020-
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: 0.035
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6s3s
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10096.png

Downloads & links

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Map

FileDownload / File: emd_10096.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of the FliPQR complex from the flagellar type 3 secretion system of Vibrio mimicus.
Voxel sizeX=Y=Z: 0.822 Å
Density
Contour LevelBy AUTHOR: 0.035 / Movie #1: 0.035
Minimum - Maximum-0.039219815 - 0.092489116
Average (Standard dev.)0.0010483998 (±0.0057757446)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 210.432 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8220.8220.822
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z210.432210.432210.432
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0390.0920.001

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Supplemental data

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Mask #1

Fileemd_10096_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_10096_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 2

Fileemd_10096_half_map_1.map
Annotationhalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1

Fileemd_10096_half_map_2.map
Annotationhalf map 1
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

EntireName: FliPQR
Components
  • Complex: FliPQR
    • 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

SupramoleculeName: FliPQR / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Vibrio mimicus CAIM 602 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightExperimental: 220 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: Vibrio mimicus CAIM 602 (bacteria)
Molecular weightTheoretical: 32.431307 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKRTQRLNLT SWLTTGLLTW LLSGMLGFAS LAFAEEPLNT GIPSTAAGAS SVTVTALEKE QGNAKTIALG SSSGGSGIPA FTMTTNPDG SEDYSINLQI LALMTMLGFL PAMVILMTSF TRIVVVMSIL RQAMGLQQTP SNQVIIGIAL FLTFFIMAPV F NQINEQAV ...String:
MKRTQRLNLT SWLTTGLLTW LLSGMLGFAS LAFAEEPLNT GIPSTAAGAS SVTVTALEKE QGNAKTIALG SSSGGSGIPA FTMTTNPDG SEDYSINLQI LALMTMLGFL PAMVILMTSF TRIVVVMSIL RQAMGLQQTP SNQVIIGIAL FLTFFIMAPV F NQINEQAV QPYLNEQISA RQAFDLAQEP MKAFMLKQTR IKDLETFVEM SGSQVTAPEQ VSMAVLIPAF ITSELKTAFQ IG FMLFLPF LIIDLVVASV LMAMGMMMLS PMIVSLPFKL MLFVLVDGWN LILSTLAGSF AL

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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: Vibrio mimicus CAIM 602 (bacteria)
Molecular weightTheoretical: 32.894637 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MEYPASVVLD FIANYFWPYT RIAAMLMVMT VTGARFVPAR VRLYLGLALT FAVMPAIPAV PSDIALLSLQ GFMITFEQIV IGMAMGMVT QFLVQIFVML GQILGMQSSL GFASMVDPAN GQNTPLLGQM FMLLATLFFL SSDGHLKMIQ LVVFSFKSLP I GSGSLTTV ...String:
MEYPASVVLD FIANYFWPYT RIAAMLMVMT VTGARFVPAR VRLYLGLALT FAVMPAIPAV PSDIALLSLQ GFMITFEQIV IGMAMGMVT QFLVQIFVML GQILGMQSSL GFASMVDPAN GQNTPLLGQM FMLLATLFFL SSDGHLKMIQ LVVFSFKSLP I GSGSLTTV DYRELALWLG IMFKASLAVS LSGIIALLTV NLSFGVMTRA APQLNIFSLG FSFALLVGLL LCWYILSGLY TH YEIYWQE TEEQICRLIR LNCENLYFQG QFGSWSHPQF EKGGGSGGGS GGGSWSHPQF EK

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Macromolecule #3: Flagellar biosynthetic protein FliQ

MacromoleculeName: Flagellar biosynthetic protein FliQ / type: protein_or_peptide / ID: 3 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Vibrio mimicus CAIM 602 (bacteria)
Molecular weightTheoretical: 10.333578 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MTPEIFVELF KESLWLVLIM VCAIIIPSLL IGLVVAIFQA ATSINEQTLS FLPRLIITLL ALMFFGHWMT QMLMDFFYSM IERLPQVLY

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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

Concentration1 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
100.0 mMC4H11NO3Tris
150.0 mMNaClSodium chloridesodium chloride
1.0 mMC10H16N2O8EDTAEthylenediaminetetraacetic acid
0.01 %C47H88O22LMNG

Details: Additional datasets were collected of the sample supplemented with 0.05, 0.5 and 3 mM fluorinated fos-choline 8
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 10 seconds wait time before blotting.
DetailsThe sample concentration was 2.7 mg/ml for datasets supplemented with fluorinated fos-choline 8.

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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
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Number grids imaged: 4 / 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: 1050955
Startup modelType of model: EMDB MAP
EMDB ID:

4173

Initial angle assignmentType: PROJECTION MATCHING
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 243489
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-6s3s:
Structure of the FliPQR complex from the flagellar type 3 secretion system of Vibrio mimicus.

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