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- EMDB-31553: Cryo-EM structure of the nonameric SsaV cytosolic domain with D9 ... -

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Basic information

Entry
Database: EMDB / ID: EMD-31553
TitleCryo-EM structure of the nonameric SsaV cytosolic domain with D9 symmetry
Map data
Sample
  • Organelle or cellular component: export gate protein of type III secretion systemType three secretion system
    • Protein or peptide: Secretion system apparatus protein SsaVSecretion
Function / homology
Function and homology information


protein secretion / plasma membrane
Similarity search - Function
Type III secretion protein HrcV / FHIPEP, domain 1 / 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
Secretion system apparatus protein SsaV
Similarity search - Component
Biological speciesSalmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.55 Å
AuthorsXu JH / Zhang YQ / Gao X
Funding support China, 1 items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China) China
CitationJournal: Microbiol Spectr / Year: 2021
Title: Structural and Functional Analysis of SsaV Cytoplasmic Domain and Variable Linker States in the Context of the InvA-SsaV Chimeric Protein.
Authors: Jinghua Xu / Jiuqing Wang / Aijun Liu / Yanqing Zhang / Xiang Gao /
Abstract: The type III secretion (T3S) injectisome is a syringe-like protein-delivery nanomachine widely utilized by Gram-negative bacteria. It can deliver effector proteins directly from bacteria into ...The type III secretion (T3S) injectisome is a syringe-like protein-delivery nanomachine widely utilized by Gram-negative bacteria. It can deliver effector proteins directly from bacteria into eukaryotic host cells, which is crucial for the bacterial-host interaction. Intracellular pathogen Salmonella enterica serovar Typhimurium encodes two sets of T3S injectisomes from Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2), which are critical for its host invasion and intracellular survival, respectively. The inner membrane export gate protein, SctV (InvA in SPI-1 and SsaV in SPI-2), is the largest component of the injectisome and is essential for assembly and function of T3SS. Here, we report the 2.11 Å cryo-EM structure of the SsaV cytoplasmic domain (SsaV) in the context of a full-length SctV chimera consisting of the transmembrane region of InvA, the linker of SsaV (SsaV) and SsaV. The structural analysis shows that SsaV exists in a semi-open state and SsaV exhibits two major orientations, implying a highly dynamic process of SsaV for the substrate selection and secretion in a full-length context. A biochemical assay indicates that SsaV plays an essential role in maintaining the nonameric state of SsaV. This study offers near atomic-level insights into how SsaV and SsaV facilitate the assembly and function of SsaV and may lead to the development of potential anti-virulence therapeutics against T3SS-mediated bacterial infection. Type III secretion system (T3SS) is a multicomponent nanomachine and a critical virulence factor for a wide range of Gram-negative bacterial pathogens. It can deliver numbers of effectors into the host cell to facilitate the bacterial host infection. Export gate protein SctV, as one of the engines of T3SS, is at the center of T3SS assembly and function. In this study, we show the high-resolution atomic structure of the cytosolic domain of SctV in the nonameric state with variable linker conformations. Our first observation of conformational changes of the linker region of SctV and the semi-open state of the cytosolic domain of SctV in the full-length context further support that the substrate selection and secretion process of SctV is highly dynamic. These findings have important implications for the development of therapeutic strategies targeting SctV to combat T3SS-mediated bacterial infection.
History
DepositionJul 19, 2021-
Header (metadata) releaseFeb 16, 2022-
Map releaseFeb 16, 2022-
UpdateFeb 16, 2022-
Current statusFeb 16, 2022Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.55
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.55
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7fed
  • Surface level: 0.55
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7fed
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_31553.png

Downloads & links

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Map

FileDownload / File: emd_31553.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.8433 Å
Density
Contour LevelBy AUTHOR: 0.55 / Movie #1: 0.55
Minimum - Maximum-2.7774744 - 3.9838123
Average (Standard dev.)0.0009957477 (±0.10994771)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 337.32 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.84330.84330.8433
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z337.320337.320337.320
α/β/γ90.00090.00090.000
start NX/NY/NZ535455
NX/NY/NZ134138134
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-2.7773.9840.001

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Supplemental data

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Sample components

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Entire : export gate protein of type III secretion system

EntireName: export gate protein of type III secretion systemType three secretion system
Components
  • Organelle or cellular component: export gate protein of type III secretion systemType three secretion system
    • Protein or peptide: Secretion system apparatus protein SsaVSecretion

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Supramolecule #1: export gate protein of type III secretion system

SupramoleculeName: export gate protein of type III secretion system / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Macromolecule #1: Secretion system apparatus protein SsaV

MacromoleculeName: Secretion system apparatus protein SsaV / type: protein_or_peptide / ID: 1 / Number of copies: 18 / Enantiomer: LEVO
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
Strain: LT2
Molecular weightTheoretical: 38.12418 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MVPGACPLIL RLSPTLHSAD LIRDIDAMRW FLFEDTGVPL PEVNIEVLPE PTEKLTVLLY QEPVFSLSIP AQADYLLIGA DASVVGDSQ TLPNGMGQIC WLTKDMAHKA QGFGLDVFAG SQRISALLKC VLLRHMGEFI GVQETRYLMN AMEKNYSELV K ELQRQLPI ...String:
MVPGACPLIL RLSPTLHSAD LIRDIDAMRW FLFEDTGVPL PEVNIEVLPE PTEKLTVLLY QEPVFSLSIP AQADYLLIGA DASVVGDSQ TLPNGMGQIC WLTKDMAHKA QGFGLDVFAG SQRISALLKC VLLRHMGEFI GVQETRYLMN AMEKNYSELV K ELQRQLPI NKIAETLQRL VSERVSIRDL RLIFGTLIDW APREKDVLML TEYVRIALRR HILRRLNPEG KPLPILRIGE GI ENLVRES IRQTAMGTYT ALSSRHKTQI LQLIEQALKQ SAKLFIVTSV DTRRFLRKIT EATLFDVPIL SWQELGEESL IQV VESIDL SEEELADNEE

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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

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Electron microscopy

MicroscopeFEI TITAN
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 K3 (6k x 4k) / Average electron dose: 50.0 e/Å2

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Image processing

Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.55 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 111741

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