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- PDB-7fec: Cryo-EM structure of the nonameric SsaV cytosolic domain with C9 ... -

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Entry
Database: PDB / ID: 7fec
TitleCryo-EM structure of the nonameric SsaV cytosolic domain with C9 symmetry
ComponentsSecretion system apparatus protein SsaVSecretion
KeywordsPROTEIN TRANSPORT / transporter
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.64 Å
AuthorsXu, J.H. / Zhang, Y.Q. / Gao, X.
Funding support China, 1items
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, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 16, 2022Provider: repository / Type: Initial release

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Assembly

Deposited unit
E: Secretion system apparatus protein SsaV
A: Secretion system apparatus protein SsaV
B: Secretion system apparatus protein SsaV
C: Secretion system apparatus protein SsaV
D: Secretion system apparatus protein SsaV
F: Secretion system apparatus protein SsaV
G: Secretion system apparatus protein SsaV
H: Secretion system apparatus protein SsaV
I: Secretion system apparatus protein SsaV


Theoretical massNumber of molelcules
Total (without water)343,1189
Polymers343,1189
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area21920 Å2
ΔGint-79 kcal/mol
Surface area138880 Å2

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Components

#1: Protein
Secretion system apparatus protein SsaV / Secretion


Mass: 38124.180 Da / Num. of mol.: 9
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
Strain: LT2 / Gene: ssaV, STM1414 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P74856

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: export gate protein of type III secretion systemType three secretion system
Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: FEI TITAN
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.64 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 90916 / Symmetry type: POINT

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