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- EMDB-20556: Focussed refinement of InvGN0N1:PrgHK:SpaPQR:PrgIJ from Salmonell... -

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Entry
Database: EMDB / ID: EMD-20556
TitleFocussed refinement of InvGN0N1:PrgHK:SpaPQR:PrgIJ from Salmonella SPI-1 injectisome NC-base
Map dataFocussed refinement of the Salmonella SPI-1 injectisome needle complex components PrgHK, SpaPQR, PrgIJ, and InvG-N0N1
Sample
  • Complex: InvGN0N1:SpaPQR:PrgHK from SPI-1 injectisome NC-base
    • Protein or peptide: Lipoprotein PrgK
    • Protein or peptide: Protein InvG
    • Protein or peptide: Protein PrgH
    • Protein or peptide: Surface presentation of antigens protein SpaP
    • Protein or peptide: Surface presentation of antigens protein SpaR
    • Protein or peptide: Surface presentation of antigens protein SpaQ
    • Protein or peptide: Protein PrgJ
    • Protein or peptide: Protein PrgI
Function / homology
Function and homology information


The IPAF inflammasome / type III protein secretion system complex / type II protein secretion system complex / protein secretion by the type III secretion system / protein secretion / protein targeting / cell outer membrane / protein transport / cell surface / extracellular region ...The IPAF inflammasome / type III protein secretion system complex / type II protein secretion system complex / protein secretion by the type III secretion system / protein secretion / protein targeting / cell outer membrane / protein transport / cell surface / extracellular region / identical protein binding / plasma membrane
Similarity search - Function
: / Type III secretion protein SpaR/YscT / Type III secretion protein HrpO / Yop virulence translocation protein R / Type III secretion system, PrgH/EprH / Type III secretion system lipoprotein HrcJ/YscJ / Type III secretion system inner membrane R protein / Bacterial export protein family 3 / Bacterial export proteins, family 1 / Bacterial export proteins, family 3 ...: / Type III secretion protein SpaR/YscT / Type III secretion protein HrpO / Yop virulence translocation protein R / Type III secretion system, PrgH/EprH / Type III secretion system lipoprotein HrcJ/YscJ / 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 / Type III secretion system, PrgH/EprH-like / FliP family / Type III secretion system protein PrgH-EprH (PrgH) / Flagella transport protein fliP family signature 2. / : / SPI-1 type 3 secretion system secretin, N0 domain / Type III secretion system outer membrane pore YscC/HrcC / Type III secretion, needle-protein-like / Type III secretion, needle-protein-like superfamily / Type III secretion needle MxiH, YscF, SsaG, EprI, PscF, EscF / Type III secretion system, needle protein / Bacterial type II secretion system protein D signature. / Type II secretion system protein GspD, conserved site / NolW-like / Bacterial type II/III secretion system short domain / NolW-like superfamily / Type II/III secretion system / Bacterial type II and III secretion system protein / 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
Surface presentation of antigens protein SpaQ / SPI-1 type 3 secretion system secretin / Surface presentation of antigens protein SpaP / Surface presentation of antigens protein SpaR / Protein PrgH / SPI-1 type 3 secretion system needle filament protein / Protein PrgJ / Lipoprotein PrgK
Similarity search - Component
Biological speciesSalmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsHu J / Worrall LJ / Strynadka NCJ
Funding support Canada, 1 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR) Canada
CitationJournal: Nat Microbiol / Year: 2019
Title: T3S injectisome needle complex structures in four distinct states reveal the basis of membrane coupling and assembly.
Authors: Jinhong Hu / Liam J Worrall / Marija Vuckovic / Chuan Hong / Wanyin Deng / Claire E Atkinson / B Brett Finlay / Zhiheng Yu / Natalie C J Strynadka /
Abstract: The bacterial injectisome is a syringe-shaped macromolecular nanomachine utilized by many pathogenic Gram-negative bacteria, including the causative agents of plague, typhoid fever, whooping cough, ...The bacterial injectisome is a syringe-shaped macromolecular nanomachine utilized by many pathogenic Gram-negative bacteria, including the causative agents of plague, typhoid fever, whooping cough, sexually transmitted infections and major nosocomial infections. Bacterial proteins destined for self-assembly and host-cell targeting are translocated by the injectisome in a process known as type III secretion (T3S). The core structure is the ~4 MDa needle complex (NC), built on a foundation of three highly oligomerized ring-forming proteins that create a hollow scaffold spanning the bacterial inner membrane (IM) (24-mer ring-forming proteins PrgH and PrgK in the Salmonella enterica serovar Typhimurium Salmonella pathogenicity island 1 (SPI-1) type III secretion system (T3SS)) and outer membrane (OM) (15-mer InvG, a member of the broadly conserved secretin pore family). An internalized helical needle projects from the NC and bacterium, ultimately forming a continuous passage to the host, for delivery of virulence effectors. Here, we have captured snapshots of the entire prototypical SPI-1 NC in four distinct needle assembly states, including near-atomic resolution, and local reconstructions in the absence and presence of the needle. These structures reveal the precise localization and molecular interactions of the internalized SpaPQR 'export apparatus' complex, which is intimately encapsulated and stabilized within the IM rings in the manner of a nanodisc, and to which the PrgJ rod directly binds and functions as an initiator and anchor of needle polymerization. We also describe the molecular details of the extensive and continuous coupling interface between the OM secretin and IM rings, which is remarkably facilitated by a localized 16-mer stoichiometry in the periplasmic-most coupling domain of the otherwise 15-mer InvG oligomer.
History
DepositionAug 2, 2019-
Header (metadata) releaseSep 18, 2019-
Map releaseOct 23, 2019-
UpdateJan 15, 2020-
Current statusJan 15, 2020Processing site: RCSB / 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-6q16
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6q16
  • 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_20556.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFocussed refinement of the Salmonella SPI-1 injectisome needle complex components PrgHK, SpaPQR, PrgIJ, and InvG-N0N1
Voxel sizeX=Y=Z: 1.71 Å
Density
Contour LevelBy AUTHOR: 0.035 / Movie #1: 0.035
Minimum - Maximum-0.07869259 - 0.14726743
Average (Standard dev.)0.00080025796 (±0.0062590195)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions250250250
Spacing250250250
CellA=B=C: 427.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.711.711.71
M x/y/z250250250
origin x/y/z0.0000.0000.000
length x/y/z427.500427.500427.500
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS250250250
D min/max/mean-0.0790.1470.001

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Supplemental data

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Sample components

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Entire : InvGN0N1:SpaPQR:PrgHK from SPI-1 injectisome NC-base

EntireName: InvGN0N1:SpaPQR:PrgHK from SPI-1 injectisome NC-base
Components
  • Complex: InvGN0N1:SpaPQR:PrgHK from SPI-1 injectisome NC-base
    • Protein or peptide: Lipoprotein PrgK
    • Protein or peptide: Protein InvG
    • Protein or peptide: Protein PrgH
    • Protein or peptide: Surface presentation of antigens protein SpaP
    • Protein or peptide: Surface presentation of antigens protein SpaR
    • Protein or peptide: Surface presentation of antigens protein SpaQ
    • Protein or peptide: Protein PrgJ
    • Protein or peptide: Protein PrgI

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Supramolecule #1: InvGN0N1:SpaPQR:PrgHK from SPI-1 injectisome NC-base

SupramoleculeName: InvGN0N1:SpaPQR:PrgHK from SPI-1 injectisome NC-base / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720) (bacteria)
Molecular weightTheoretical: 1.5 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 typhimurium (strain LT2 / SGSC1412 / ATCC 700720) (bacteria)
Strain: LT2 / SGSC1412 / ATCC 700720
Molecular weightTheoretical: 28.245287 KDa
SequenceString: MIRRYLYTFL LVMTLAGCKD KDLLKGLDQE QANEVIAVLQ MHNIEANKID SGKLGYSITV AEPDFTAAVY WIKTYQLPPR PRVEIAQMF PADSLVSSPR AEKARLYSAI EQRLEQSLQT MEGVLSARVH ISYDIDAGEN GRPPKPVHLS ALAVYERGSP L AHQISDIK ...String:
MIRRYLYTFL LVMTLAGCKD KDLLKGLDQE QANEVIAVLQ MHNIEANKID SGKLGYSITV AEPDFTAAVY WIKTYQLPPR PRVEIAQMF PADSLVSSPR AEKARLYSAI EQRLEQSLQT MEGVLSARVH ISYDIDAGEN GRPPKPVHLS ALAVYERGSP L AHQISDIK RFLKNSFADV DYDNISVVLS ERSDAQLQAP GTPVKRNSFA TSWIVLIILL SVMSAGFGVW YYKNHYARNK KG ITADDKA KSSNE

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Macromolecule #2: Protein InvG

MacromoleculeName: Protein InvG / type: protein_or_peptide / ID: 2 / Number of copies: 16 / Enantiomer: LEVO
Source (natural)Organism: Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720) (bacteria)
Strain: LT2 / SGSC1412 / ATCC 700720
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

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Macromolecule #3: Protein PrgH

MacromoleculeName: Protein PrgH / type: protein_or_peptide / ID: 3 / Number of copies: 24 / Enantiomer: LEVO
Source (natural)Organism: Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720) (bacteria)
Strain: LT2 / SGSC1412 / ATCC 700720
Molecular weightTheoretical: 44.509367 KDa
SequenceString: METSKEKTIT SPGPYIVRLL NSSLNGCEFP LLTGRTLFVV GQSDALTASG QLPDIPADSF FIPLDHGGVN FEIQVDTDAT EIILHELKE GNSESRSVQL NTPIQVGELL ILIRPESEPW VPEQPEKLET SAKKNEPRFK NGIVAALAGF FILGIGTVGT L WILNSPQR ...String:
METSKEKTIT SPGPYIVRLL NSSLNGCEFP LLTGRTLFVV GQSDALTASG QLPDIPADSF FIPLDHGGVN FEIQVDTDAT EIILHELKE GNSESRSVQL NTPIQVGELL ILIRPESEPW VPEQPEKLET SAKKNEPRFK NGIVAALAGF FILGIGTVGT L WILNSPQR QAAELDSLLG QEKERFQVLP GRDKMLYVAA QNERDTLWAR QVLARGDYDK NARVINENEE NKRISIWLDT YY PQLAYYR IHFDEPRKPV FWLSRQRNTM SKKELEVLSQ KLRALMPYAD SVNITLMDDV TAAGQAEAGL KQQALPYSRR NHK GGVTFV IQGALDDVEI LRARQFVDSY YRTWGGRYVQ FAIELKDDWL KGRSFQYGAE GYIKMSPGHW YFPSPL

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Macromolecule #4: Surface presentation of antigens protein SpaP

MacromoleculeName: Surface presentation of antigens protein SpaP / type: protein_or_peptide / ID: 4 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720) (bacteria)
Strain: LT2 / SGSC1412 / ATCC 700720
Molecular weightTheoretical: 25.249596 KDa
SequenceString: MGNDISLIAL LAFSTLLPFI IASGTCFVKF SIVFVMVRNA LGLQQIPSNM TLNGVALLLS MFVMWPIMHD AYVYFEDEDV TFNDISSLS KHVDEGLDGY RDYLIKYSDR ELVQFFENAQ LKRQYGEETE TVKRDKDEIE KPSIFALLPA YALSEIKSAF K IGFYLYLP ...String:
MGNDISLIAL LAFSTLLPFI IASGTCFVKF SIVFVMVRNA LGLQQIPSNM TLNGVALLLS MFVMWPIMHD AYVYFEDEDV TFNDISSLS KHVDEGLDGY RDYLIKYSDR ELVQFFENAQ LKRQYGEETE TVKRDKDEIE KPSIFALLPA YALSEIKSAF K IGFYLYLP FVVVDLVVSS VLLALGMMMM SPVTISTPIK LVLFVALDGW TLLSKGLILQ YMDIAT

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Macromolecule #5: Surface presentation of antigens protein SpaR

MacromoleculeName: Surface presentation of antigens protein SpaR / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720) (bacteria)
Strain: LT2 / SGSC1412 / ATCC 700720
Molecular weightTheoretical: 28.499533 KDa
SequenceString: MFYALYFEIH HLVASAALGF ARVAPIFFFL PFLNSGVLSG APRNAIIILV ALGVWPHALN EAPPFLSVAM IPLVLQEAAV GVMLGCLLS WPFWVMHALG CIIDNQRGAT LSSSIDPANG IDTSEMANFL NMFAAVVYLQ NGGLVTMVDV LNKSYQLCDP M NECTPSLP ...String:
MFYALYFEIH HLVASAALGF ARVAPIFFFL PFLNSGVLSG APRNAIIILV ALGVWPHALN EAPPFLSVAM IPLVLQEAAV GVMLGCLLS WPFWVMHALG CIIDNQRGAT LSSSIDPANG IDTSEMANFL NMFAAVVYLQ NGGLVTMVDV LNKSYQLCDP M NECTPSLP PLLTFINQVA QNALVLASPV VLVLLLSEVF LGLLSRFAPQ MNAFAISLTV KSGIAVLIML LYFSPVLPDN VL RLSFQAT GLSSWFYERG ATHVLE

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Macromolecule #6: Surface presentation of antigens protein SpaQ

MacromoleculeName: Surface presentation of antigens protein SpaQ / type: protein_or_peptide / ID: 6 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720) (bacteria)
Strain: LT2 / SGSC1412 / ATCC 700720
Molecular weightTheoretical: 9.363229 KDa
SequenceString:
MDDLVFAGNK ALYLVLILSG WPTIVATIIG LLVGLFQTVT QLQEQTLPFG IKLLGVCLCL FLLSGWYGEV LLSYGRQVIF LALAKG

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Macromolecule #7: Protein PrgJ

MacromoleculeName: Protein PrgJ / type: protein_or_peptide / ID: 7 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720) (bacteria)
Strain: LT2 / SGSC1412 / ATCC 700720
Molecular weightTheoretical: 10.934425 KDa
SequenceString:
MSIATIVPEN AVIGQAVNIR SMETDIVSLD DRLLQAFSGS AIATAVDKQT ITNRIEDPNL VTDPKELAIS QEMISDYNLY VSMVSTLTR KGVGAVETLL RS

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Macromolecule #8: Protein PrgI

MacromoleculeName: Protein PrgI / type: protein_or_peptide / ID: 8 / Number of copies: 13 / Enantiomer: LEVO
Source (natural)Organism: Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720) (bacteria)
Strain: LT2 / SGSC1412 / ATCC 700720
Molecular weightTheoretical: 8.864868 KDa
SequenceString:
MATPWSGYLD DVSAKFDTGV DNLQTQVTEA LDKLAAKPSD PALLAAYQSK LSEYNLYRNA QSNTVKVFKD IDAAIIQNFR

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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

BufferpH: 8
GridDetails: unspecified
VitrificationCryogen name: ETHANE

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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: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 51.3 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 19141

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