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- EMDB-8398: Single particle cryo-EM reconstruction of the Salmonella SPI-1 ty... -

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Basic information

Entry
Database: EMDB / ID: EMD-8398
TitleSingle particle cryo-EM reconstruction of the Salmonella SPI-1 type III secretion injectisome basal body at 4.3 Angstrom resolutionType three secretion system
Map data24-fold averaged map of the basal body complex
Sample
  • Complex: Type III secretion injectisome basal bodyType three secretion system
    • Protein or peptide: Lipoprotein PrgK
    • Protein or peptide: Protein PrgH
KeywordsBacterial / secretion / injectisome / membrane protein
Function / homology
Function and homology information


protein secretion / cell outer membrane / plasma membrane
Similarity search - Function
Type III secretion system, PrgH/EprH / Type III secretion system lipoprotein HrcJ/YscJ / Type III secretion system, PrgH/EprH-like / Type III secretion system protein PrgH-EprH (PrgH) / Lipoprotein YscJ/Flagellar M-ring protein / Secretory protein of YscJ/FliF family / Flagellar M-ring , N-terminal / AMP-binding enzyme, C-terminal domain superfamily / Prokaryotic membrane lipoprotein lipid attachment site profile.
Similarity search - Domain/homology
Protein PrgH / Lipoprotein PrgK
Similarity search - Component
Biological speciesSalmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsWorrall LJ / Hong C
Funding support Canada, 1 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR) Canada
CitationJournal: Nature / Year: 2016
Title: Near-atomic-resolution cryo-EM analysis of the Salmonella T3S injectisome basal body.
Authors: L J Worrall / C Hong / M Vuckovic / W Deng / J R C Bergeron / D D Majewski / R K Huang / T Spreter / B B Finlay / Z Yu / N C J Strynadka /
Abstract: The type III secretion (T3S) injectisome is a specialized protein nanomachine that is critical for the pathogenicity of many Gram-negative bacteria, including purveyors of plague, typhoid fever, ...The type III secretion (T3S) injectisome is a specialized protein nanomachine that is critical for the pathogenicity of many Gram-negative bacteria, including purveyors of plague, typhoid fever, whooping cough, sexually transmitted infections and major nosocomial infections. This syringe-shaped 3.5-MDa macromolecular assembly spans both bacterial membranes and that of the infected host cell. The internal channel formed by the injectisome allows for the direct delivery of partially unfolded virulence effectors into the host cytoplasm. The structural foundation of the injectisome is the basal body, a molecular lock-nut structure composed predominantly of three proteins that form highly oligomerized concentric rings spanning the inner and outer membranes. Here we present the structure of the prototypical Salmonella enterica serovar Typhimurium pathogenicity island 1 basal body, determined using single-particle cryo-electron microscopy, with the inner-membrane-ring and outer-membrane-ring oligomers defined at 4.3 Å and 3.6 Å resolution, respectively. This work presents the first, to our knowledge, high-resolution structural characterization of the major components of the basal body in the assembled state, including that of the widespread class of outer-membrane portals known as secretins.
History
DepositionSep 15, 2016-
Header (metadata) releaseNov 9, 2016-
Map releaseDec 21, 2016-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5tcp
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5tcp
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5tcp
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_8398.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation24-fold averaged map of the basal body complex
Voxel sizeX=Y=Z: 1.71 Å
Density
Contour LevelBy AUTHOR: 0.06 / Movie #1: 0.06
Minimum - Maximum-0.2226074 - 0.2467463
Average (Standard dev.)0.0002957704 (±0.011063291)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 513.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.711.711.71
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z513.000513.000513.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-152-37
NX/NY/NZ998271
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.2230.2470.000

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Supplemental data

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Sample components

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Entire : Type III secretion injectisome basal body

EntireName: Type III secretion injectisome basal bodyType three secretion system
Components
  • Complex: Type III secretion injectisome basal bodyType three secretion system
    • Protein or peptide: Lipoprotein PrgK
    • Protein or peptide: Protein PrgH

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Supramolecule #1: Type III secretion injectisome basal body

SupramoleculeName: Type III secretion injectisome basal body / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: PrgH130-392 mutant
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Molecular weightTheoretical: 2 MDa

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Macromolecule #1: Lipoprotein PrgK

MacromoleculeName: Lipoprotein PrgK / type: protein_or_peptide / ID: 1 / Number of copies: 24 / Enantiomer: LEVO
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Molecular weightTheoretical: 26.199723 KDa
SequenceString: CKDKDLLKGL DQEQANEVIA VLQMHNIEAN KIDSGKLGYS ITVAEPDFTA AVYWIKTYQL PPRPRVEIAQ MFPADSLVSS PRAEKARLY SAIEQRLEQS LQTMEGVLSA RVHISYDIDA GENGRPPKPV HLSALAVYER GSPLAHQISD IKRFLKNSFA D VDYDNISV ...String:
CKDKDLLKGL DQEQANEVIA VLQMHNIEAN KIDSGKLGYS ITVAEPDFTA AVYWIKTYQL PPRPRVEIAQ MFPADSLVSS PRAEKARLY SAIEQRLEQS LQTMEGVLSA RVHISYDIDA GENGRPPKPV HLSALAVYER GSPLAHQISD IKRFLKNSFA D VDYDNISV VLSERSDAQL QAPGTPVKRN SFATSWIVLI ILLSVMSAGF GVWYYKNHYA RNKKGITADD KAKSSNE

UniProtKB: Lipoprotein PrgK

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Macromolecule #2: Protein PrgH

MacromoleculeName: Protein PrgH / type: protein_or_peptide / ID: 2 / Number of copies: 24 / Enantiomer: LEVO
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Molecular weightTheoretical: 30.360537 KDa
SequenceString: SAKKNEPRFK NGIVAALAGF FILGIGTVGT LWILNSPQRQ AAELDSLLGQ EKERFQVLPG RDKMLYVAAQ NERDTLWARQ VLARGDYDK NARVINENEE NKRISIWLDT YYPQLAYYRI HFDEPRKPVF WLSRQRNTMS KKELEVLSQK LRALMPYADS V NITLMDDV ...String:
SAKKNEPRFK NGIVAALAGF FILGIGTVGT LWILNSPQRQ AAELDSLLGQ EKERFQVLPG RDKMLYVAAQ NERDTLWARQ VLARGDYDK NARVINENEE NKRISIWLDT YYPQLAYYRI HFDEPRKPVF WLSRQRNTMS KKELEVLSQK LRALMPYADS V NITLMDDV TAAGQAEAGL KQQALPYSRR NHKGGVTFVI QGALDDVEIL RARQFVDSYY RTWGGRYVQF AIELKDDWLK GR SFQYGAE GYIKMSPGHW YFPSPL

UniProtKB: Protein PrgH

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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

Concentration10 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
10.0 mMC4H11NO3Tris
500.0 mMNaClSodium chloridesodium chloride
0.2 %LDAO
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.038 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 298 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 opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 3.2 µm / Calibrated defocus min: 1.3 µm / Calibrated magnification: 29240 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.01 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 64000
Specialist opticsEnergy filter - Name: Gatan GIF / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 80.0 K / Max: 80.0 K
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7676 pixel / Digitization - Dimensions - Height: 7420 pixel / Digitization - Frames/image: 1-48 / Number grids imaged: 1 / Number real images: 2515 / Average exposure time: 0.375 sec. / Average electron dose: 1.3 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 263900
Startup modelType of model: OTHER / Details: e2initialmodel.py
Initial angle assignmentType: OTHER / Software: (Name: RELION, FREALIGN)
Final angle assignmentType: OTHER / Software - Name: FREALIGN
Final reconstructionApplied symmetry - Point group: C24 (24 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN / Details: Limited frequency refinement in Frealign / Number images used: 67800

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Atomic model buiding 1

DetailsInitial fitting was carried out with Chimera, followed by rebuilding and refinement in Rosetta, Phenix, and Coot.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-5tcp:
Near-atomic resolution cryo-EM structure of the periplasmic domains of PrgH and PrgK

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