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- EMDB-22589: Cryo-EM structure of the nonameric EscV cytosolic domain from the... -

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Basic information

Entry
Database: EMDB / ID: EMD-22589
TitleCryo-EM structure of the nonameric EscV cytosolic domain from the type III secretion system
Map data
Sample
  • Complex: EscV Cytosolic Nonamer Ring
    • Protein or peptide: Translocator EscV
KeywordsInjectisome / Nonamer / Export Apparatus / Secretion / PROTEIN TRANSPORT
Function / homology
Function and homology information


protein secretion / plasma membrane
Similarity search - Function
Type III secretion protein HrcV / FHIPEP conserved site / Bacterial export FHIPEP family signature. / Type III secretion system FHIPEP / FHIPEP, domain 3 / FHIPEP, domain 4 / FHIPEP family
Similarity search - Domain/homology
Biological speciesEscherichia coli O127:H6 str. E2348/69 (bacteria) / Escherichia coli O127:H6 (strain E2348/69 / EPEC) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.7 Å
AuthorsMajewski DD / Lyons BJE
Funding support Canada, 3 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR) Canada
Natural Sciences and Engineering Research Council (NSERC, Canada) Canada
Howard Hughes Medical Institute (HHMI) Canada
CitationJournal: J Struct Biol / Year: 2020
Title: Cryo-EM analysis of the SctV cytosolic domain from the enteropathogenic E. coli T3SS injectisome.
Authors: Dorothy D Majewski / Bronwyn J E Lyons / Claire E Atkinson / Natalie C J Strynadka /
Abstract: The bacterial injectisome and flagella both rely on type III secretion systems for their assembly. The syringe-like injectisome creates a continuous channel between the bacterium and the host cell, ...The bacterial injectisome and flagella both rely on type III secretion systems for their assembly. The syringe-like injectisome creates a continuous channel between the bacterium and the host cell, through which signal-modulating effector proteins are secreted. The inner membrane pore protein SctV controls the hierarchy of substrate selection and may also be involved in energizing secretion. We present the 4.7 Å cryo-EM structure of the SctV cytosolic domain (SctV) from the enteropathogenic Escherichia coli injectisome. SctV forms a nonameric ring with primarily electrostatic interactions between its subunits. Molecular dynamics simulations show that monomeric SctV maintains a closed conformation, in contrast with previous studies on flagellar homologue FlhA. Comparison with substrate-bound homologues suggest that a conformational change would be required to accommodate binding partners.
History
DepositionSep 3, 2020-
Header (metadata) releaseNov 25, 2020-
Map releaseNov 25, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.017
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.017
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7k08
  • Surface level: 0.017
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7k08
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22589.png

Downloads & links

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Map

FileDownload / File: emd_22589.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.852 Å
Density
Contour LevelBy AUTHOR: 0.017 / Movie #1: 0.017
Minimum - Maximum-0.029099936 - 0.06602799
Average (Standard dev.)0.00031795565 (±0.0027968623)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 272.64 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8520.8520.852
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z272.640272.640272.640
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ256256256
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0290.0660.000

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Supplemental data

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Sample components

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Entire : EscV Cytosolic Nonamer Ring

EntireName: EscV Cytosolic Nonamer Ring
Components
  • Complex: EscV Cytosolic Nonamer Ring
    • Protein or peptide: Translocator EscV

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Supramolecule #1: EscV Cytosolic Nonamer Ring

SupramoleculeName: EscV Cytosolic Nonamer Ring / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli O127:H6 str. E2348/69 (bacteria)
Molecular weightTheoretical: 350 KDa

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Macromolecule #1: Translocator EscV

MacromoleculeName: Translocator EscV / type: protein_or_peptide / ID: 1 / Number of copies: 9 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli O127:H6 (strain E2348/69 / EPEC) (bacteria)
Strain: E2348/69 / EPEC
Molecular weightTheoretical: 39.081633 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GSHMADLSNS QNISPGAEPL ILNLSSNIYS SDITQQIEVM RWNFFEESGI PLPKIIVNPV KNNDSAIEFL LYQESIYKDT LIDDTVYFE AGHAEISFEF VQEKLSTNSI VYKTNKTNQQ LAHLTGMDVY ATTNDKITFL LKKLVLSNAK EFIGVQETRY L MDIMERKY ...String:
GSHMADLSNS QNISPGAEPL ILNLSSNIYS SDITQQIEVM RWNFFEESGI PLPKIIVNPV KNNDSAIEFL LYQESIYKDT LIDDTVYFE AGHAEISFEF VQEKLSTNSI VYKTNKTNQQ LAHLTGMDVY ATTNDKITFL LKKLVLSNAK EFIGVQETRY L MDIMERKY NELVKELQRQ LGLSKIVDIL QRLVEENVSI RDLRTIFETL IFWSTKEKDV VILCEYVRIA LRRHILGRYS VS GTLLNVW LIGSDIENEL RESIRQTSSG SYLNISPERT EQIIGFLKNI MNPTGNGVIL TALDIRRYVK KMIEGSFPSV PVL SFQEVG NNIELKVLGT VNDFRA

UniProtKB: Translocator EscV

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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

Concentration1.5 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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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
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C9 (9 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 83566
FSC plot (resolution estimation)

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