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- EMDB-8399: Single particle cryo-EM reconstruction of the Salmonella SPI-1 ty... -

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Basic information

Entry
Database: EMDB / ID: EMD-8399
TitleSingle particle cryo-EM reconstruction of the Salmonella SPI-1 type III secretion injectisome secretin InvG at 3.6 Angstrom resolution
Map data15x symmetry averaged single particle cryo-EM reconstruction of the Salmonella type III secretion system secretin InvG at 3.6 Angstrom resolution
Sample
  • Complex: Type III injectisome basal body
    • Protein or peptide: Protein InvG
KeywordsBacterial / secretion / injectisome / membrane protein
Function / homology
Function and homology information


type III protein secretion system complex / type II protein secretion system complex / protein secretion by the type III secretion system / protein secretion / cell outer membrane / identical protein binding
Similarity search - Function
: / SPI-1 type 3 secretion system secretin, N0 domain / Type III secretion system outer membrane pore YscC/HrcC / Bacterial type II secretion system protein D signature. / Type II secretion system protein GspD, conserved site / : / NolW-like / NolW-like superfamily / Bacterial type II/III secretion system short domain / Type II/III secretion system / Bacterial type II and III secretion system protein
Similarity search - Domain/homology
SPI-1 type 3 secretion system secretin
Similarity search - Component
Biological speciesSalmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 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: 0.08
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5tcq
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5tcq
  • Surface level: 0.08
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8399.png

Downloads & links

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Map

FileDownload / File: emd_8399.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation15x symmetry averaged single particle cryo-EM reconstruction of the Salmonella type III secretion system secretin InvG at 3.6 Angstrom resolution
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.71 Å/pix.
x 300 pix.
= 513. Å		1.71 Å/pix.
x 300 pix.
= 513. Å		1.71 Å/pix.
x 300 pix.
= 513. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.71 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.08 / Movie #1: 0.08
Minimum - Maximum-0.18196891 - 0.3536772
Average (Standard dev.)0.0008786313 (±0.0072768456)
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-0.1820.3540.001

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Supplemental data

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Sample components

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Entire : Type III injectisome basal body

EntireName: Type III injectisome basal body
Components
  • Complex: Type III injectisome basal body
    • Protein or peptide: Protein InvG

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Supramolecule #1: Type III injectisome basal body

SupramoleculeName: Type III 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: Protein InvG

MacromoleculeName: Protein InvG / type: protein_or_peptide / ID: 1 / Number of copies: 15 / Enantiomer: LEVO
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium (bacteria)
Molecular weightTheoretical: 61.835559 KDa
SequenceString: MKTHILLARV LACAALVLVT PGYSSEKIPV TGSGFVAKDD SLRTFFDAMA LQLKEPVIVS KMAARKKITG NFEFHDPNAL LEKLSLQLG LIWYFDGQAI YIYDASEMRN AVVSLRNVSL NEFNNFLKRS GLYNKNYPLR GDNRKGTFYV SGPPVYVDMV V NAATMMDK ...String:
MKTHILLARV LACAALVLVT PGYSSEKIPV TGSGFVAKDD SLRTFFDAMA LQLKEPVIVS KMAARKKITG NFEFHDPNAL LEKLSLQLG LIWYFDGQAI YIYDASEMRN AVVSLRNVSL NEFNNFLKRS GLYNKNYPLR GDNRKGTFYV SGPPVYVDMV V NAATMMDK QNDGIELGRQ KIGVMRLNNT FVGDRTYNLR DQKMVIPGIA TAIERLLQGE EQPLGNIVSS EPPAMPAFSA NG EKGKAAN YAGGMSLQEA LKQNAAAGNI KIVAYPDTNS LLVKGTAEQV HFIEMLVKAL DVAKRHVELS LWIVDLNKSD LER LGTSWS GSITIGDKLG VSLNQSSIST LDGSRFIAAV NALEEKKQAT VVSRPVLLTQ ENVPAIFDNN RTFYTKLIGE RNVA LEHVT YGTMIRVLPR FSADGQIEMS LDIEDGNDKT PQSDTTTSVD ALPEVGRTLI STIARVPHGK SLLVGGYTRD ANTDT VQSI PFLGKLPLIG SLFRYSSKNK SNVVRVFMIE PKEIVDPLTP DASESVNNIL KQSGAWSGDD KLQKWVRVYL DRGQEA IK

UniProtKB: SPI-1 type 3 secretion system secretin

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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 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
TemperatureMin: 80.0 K / Max: 80.0 K
Specialist opticsEnergy filter - Name: Gatan GIF / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
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: 2685 / Average exposure time: 0.375 sec. / Average electron dose: 1.3 e/Å2
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 FIELD / Cs: 0.01 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 64000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 164300
Startup modelType of model: OTHER / Details: e2initialmodel.py
Final reconstructionApplied symmetry - Point group: C15 (15 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 83900
Initial angle assignmentType: OTHER / Software: (Name: RELION, FREALIGN)
Final angle assignmentType: OTHER / Software - Name: RELION

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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-5tcq:
Near-atomic resolution cryo-EM structure of the Salmonella SPI-1 type III secretion injectisome secretin InvG

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