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- EMDB-11781: Substrate-engaged type 3 secretion system needle complex from Sal... -

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Basic information

Entry
Database: EMDB / ID: EMD-11781
TitleSubstrate-engaged type 3 secretion system needle complex from Salmonella enterica typhimurium - SpaR state 1
Map data
Sample
  • Complex: Substrate-engaged T3SS needle complex
    • Protein or peptide: x 9 types
  • Ligand: x 2 types
KeywordsT3SS / Export Apparatus / Injectisome / Needle Complex / PROTEIN TRANSPORT / Substrate
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, PrgH/EprH-like / Type III secretion system protein PrgH-EprH (PrgH) / : / SPI-1 type 3 secretion system secretin, N0 domain ...: / 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, PrgH/EprH-like / Type III secretion system protein PrgH-EprH (PrgH) / : / 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 / 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. / 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 / 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 enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsMarlovits T
Funding support Austria, Germany, 2 items
OrganizationGrant numberCountry
Austrian Science FundI 2408-B22 Austria
German Research Foundation (DFG)FA1518/2-1 Germany
CitationJournal: Nat Commun / Year: 2021
Title: Substrate-engaged type III secretion system structures reveal gating mechanism for unfolded protein translocation.
Authors: Sean Miletic / Dirk Fahrenkamp / Nikolaus Goessweiner-Mohr / Jiri Wald / Maurice Pantel / Oliver Vesper / Vadim Kotov / Thomas C Marlovits /
Abstract: Many bacterial pathogens rely on virulent type III secretion systems (T3SSs) or injectisomes to translocate effector proteins in order to establish infection. The central component of the injectisome ...Many bacterial pathogens rely on virulent type III secretion systems (T3SSs) or injectisomes to translocate effector proteins in order to establish infection. The central component of the injectisome is the needle complex which assembles a continuous conduit crossing the bacterial envelope and the host cell membrane to mediate effector protein translocation. However, the molecular principles underlying type III secretion remain elusive. Here, we report a structure of an active Salmonella enterica serovar Typhimurium needle complex engaged with the effector protein SptP in two functional states, revealing the complete 800Å-long secretion conduit and unraveling the critical role of the export apparatus (EA) subcomplex in type III secretion. Unfolded substrates enter the EA through a hydrophilic constriction formed by SpaQ proteins, which enables side chain-independent substrate transport. Above, a methionine gasket formed by SpaP proteins functions as a gate that dilates to accommodate substrates while preventing leaky pore formation. Following gate penetration, a moveable SpaR loop first folds up to then support substrate transport. Together, these findings establish the molecular basis for substrate translocation through T3SSs and improve our understanding of bacterial pathogenicity and motility.
History
DepositionSep 24, 2020-
Header (metadata) releaseMar 17, 2021-
Map releaseMar 17, 2021-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.013
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.013
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  • Surface view with fitted model
  • Atomic models: PDB-7ah9
  • Surface level: 0.013
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ahi
  • Surface level: 0.013
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7ah9
  • Imaged by Jmol
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7ahi
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11781.png

Downloads & links

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Map

FileDownload / File: emd_11781.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 600 pix.
= 660. Å		1.1 Å/pix.
x 600 pix.
= 660. Å		1.1 Å/pix.
x 600 pix.
= 660. Å

Surface

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.013 / Movie #1: 0.013
Minimum - Maximum-0.01733566 - 0.06664831
Average (Standard dev.)0.000043450727 (±0.0027255516)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions600600600
Spacing600600600
CellA=B=C: 660.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z600600600
origin x/y/z0.0000.0000.000
length x/y/z660.000660.000660.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS600600600
D min/max/mean-0.0170.0670.000

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Supplemental data

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Additional map: postprocess Bfactor -30

Fileemd_11781_additional_1.map
Annotationpostprocess Bfactor -30
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Additional map: postprocess Bfactor -60

Fileemd_11781_additional_2.map
Annotationpostprocess Bfactor -60
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_11781_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: Refine3D

Fileemd_11781_half_map_2.map
AnnotationRefine3D
Projections & Slices
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Sample components

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Entire : Substrate-engaged T3SS needle complex

EntireName: Substrate-engaged T3SS needle complex
Components
  • Complex: Substrate-engaged T3SS needle complex
    • 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: SptP3x-GFP-FLAG
    • Protein or peptide: Protein PrgI
    • Protein or peptide: Type 3 secretion system secretin
    • Protein or peptide: Lipoprotein PrgK
    • Protein or peptide: Protein PrgH
  • Ligand: 1,2-DIACYL-GLYCEROL-3-SN-PHOSPHATE
  • Ligand: LAURYL DIMETHYLAMINE-N-OXIDE

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Supramolecule #1: Substrate-engaged T3SS needle complex

SupramoleculeName: Substrate-engaged T3SS needle complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#9
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
Molecular weightTheoretical: 2.84 MDa

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Macromolecule #1: Surface presentation of antigens protein SpaP

MacromoleculeName: Surface presentation of antigens protein SpaP / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
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

UniProtKB: Surface presentation of antigens protein SpaP

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Macromolecule #2: Surface presentation of antigens protein SpaR

MacromoleculeName: Surface presentation of antigens protein SpaR / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
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

UniProtKB: Surface presentation of antigens protein SpaR

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Macromolecule #3: Surface presentation of antigens protein SpaQ

MacromoleculeName: Surface presentation of antigens protein SpaQ / type: protein_or_peptide / ID: 3 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
Molecular weightTheoretical: 9.363229 KDa
SequenceString:
MDDLVFAGNK ALYLVLILSG WPTIVATIIG LLVGLFQTVT QLQEQTLPFG IKLLGVCLCL FLLSGWYGEV LLSYGRQVIF LALAKG

UniProtKB: Surface presentation of antigens protein SpaQ

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Macromolecule #4: Protein PrgJ

MacromoleculeName: Protein PrgJ / type: protein_or_peptide / ID: 4 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
Molecular weightTheoretical: 10.934425 KDa
SequenceString:
MSIATIVPEN AVIGQAVNIR SMETDIVSLD DRLLQAFSGS AIATAVDKQT ITNRIEDPNL VTDPKELAIS QEMISDYNLY VSMVSTLTR KGVGAVETLL RS

UniProtKB: Protein PrgJ

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Macromolecule #5: SptP3x-GFP-FLAG

MacromoleculeName: SptP3x-GFP-FLAG / type: protein_or_peptide / ID: 5
Details: SptP3xGFP sequence modeled as poly-alanine and named as unknown (UNK) in the coordinate file
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
Molecular weightTheoretical: 12.017806 KDa
Recombinant expressionOrganism: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
SequenceString: (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) ...String:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)

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Macromolecule #6: Protein PrgI

MacromoleculeName: Protein PrgI / type: protein_or_peptide / ID: 6 / Number of copies: 72 / Enantiomer: LEVO
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
Molecular weightTheoretical: 8.864868 KDa
SequenceString:
MATPWSGYLD DVSAKFDTGV DNLQTQVTEA LDKLAAKPSD PALLAAYQSK LSEYNLYRNA QSNTVKVFKD IDAAIIQNFR

UniProtKB: SPI-1 type 3 secretion system needle filament protein

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Macromolecule #7: Type 3 secretion system secretin

MacromoleculeName: Type 3 secretion system secretin / type: protein_or_peptide / ID: 7 / Number of copies: 16 / Enantiomer: LEVO
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (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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Macromolecule #8: Lipoprotein PrgK

MacromoleculeName: Lipoprotein PrgK / type: protein_or_peptide / ID: 8 / Number of copies: 24 / Enantiomer: LEVO
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
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

UniProtKB: Lipoprotein PrgK

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Macromolecule #9: Protein PrgH

MacromoleculeName: Protein PrgH / type: protein_or_peptide / ID: 9 / Number of copies: 24 / Enantiomer: LEVO
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
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

UniProtKB: Protein PrgH

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Macromolecule #10: 1,2-DIACYL-GLYCEROL-3-SN-PHOSPHATE

MacromoleculeName: 1,2-DIACYL-GLYCEROL-3-SN-PHOSPHATE / type: ligand / ID: 10 / Number of copies: 5 / Formula: 3PH
Molecular weightTheoretical: 704.998 Da
Chemical component information

ChemComp-3PH:
1,2-DIACYL-GLYCEROL-3-SN-PHOSPHATE

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Macromolecule #11: LAURYL DIMETHYLAMINE-N-OXIDE

MacromoleculeName: LAURYL DIMETHYLAMINE-N-OXIDE / type: ligand / ID: 11 / Number of copies: 9 / Formula: LDA
Molecular weightTheoretical: 229.402 Da
Chemical component information

ChemComp-LDA:
LAURYL DIMETHYLAMINE-N-OXIDE / LDAO, detergent*YM

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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
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 53.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 77411
Initial angle assignmentType: OTHER / Details: Rotationally averaged 3D structure
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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