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- PDB-7awa: SctV (SsaV) cytoplasmic domain -

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Basic information

Entry
Database: PDB / ID: 7awa
TitleSctV (SsaV) cytoplasmic domain
ComponentsSecretion system apparatus protein SsaVSecretion
KeywordsPROTEIN TRANSPORT / Type III secretion / T3SS / Export Apparatus
Function / homologyType III secretion system FHIPEP / Type III secretion protein HrcV / FHIPEP conserved site / FHIPEP, domain 3 / FHIPEP, domain 4 / protein secretion / integral component of membrane / plasma membrane / Secretion system apparatus protein SsaV
Function and homology information
Biological speciesSalmonella typhimurium (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsMatthews-Palmer, T.R.S. / Gonzalez-Rodriguez, N. / Calcraft, T. / Lagercrantz, S. / Zachs, T. / Yu, X.J. / Grabe, G. / Holden, D. / Nans, A. / Rosenthal, P. ...Matthews-Palmer, T.R.S. / Gonzalez-Rodriguez, N. / Calcraft, T. / Lagercrantz, S. / Zachs, T. / Yu, X.J. / Grabe, G. / Holden, D. / Nans, A. / Rosenthal, P. / Rouse, S. / Beeby, M.
Funding support United Kingdom, 4items
OrganizationGrant numberCountry
Cancer Research UKFC001179 United Kingdom
Medical Research Council (MRC, United Kingdom)FC001179 United Kingdom
Wellcome TrustFC001179 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/P019374/1 United Kingdom
CitationJournal: J Struct Biol / Year: 2021
Title: Structure of the cytoplasmic domain of SctV (SsaV) from the Salmonella SPI-2 injectisome and implications for a pH sensing mechanism.
Authors: Teige R S Matthews-Palmer / Nayim Gonzalez-Rodriguez / Thomas Calcraft / Signe Lagercrantz / Tobias Zachs / Xiu-Jun Yu / Grzegorz J Grabe / David W Holden / Andrea Nans / Peter B Rosenthal / ...Authors: Teige R S Matthews-Palmer / Nayim Gonzalez-Rodriguez / Thomas Calcraft / Signe Lagercrantz / Tobias Zachs / Xiu-Jun Yu / Grzegorz J Grabe / David W Holden / Andrea Nans / Peter B Rosenthal / Sarah L Rouse / Morgan Beeby /
Abstract: Bacterial type III secretion systems assemble the axial structures of both injectisomes and flagella. Injectisome type III secretion systems subsequently secrete effector proteins through their ...Bacterial type III secretion systems assemble the axial structures of both injectisomes and flagella. Injectisome type III secretion systems subsequently secrete effector proteins through their hollow needle into a host, requiring co-ordination. In the Salmonella enterica serovar Typhimurium SPI-2 injectisome, this switch is triggered by sensing the neutral pH of the host cytoplasm. Central to specificity switching is a nonameric SctV protein with an N-terminal transmembrane domain and a toroidal C-terminal cytoplasmic domain. A 'gatekeeper' complex interacts with the SctV cytoplasmic domain in a pH dependent manner, facilitating translocon secretion while repressing effector secretion through a poorly understood mechanism. To better understand the role of SctV in SPI-2 translocon-effector specificity switching, we purified full-length SctV and determined its toroidal cytoplasmic region's structure using cryo-EM. Structural comparisons and molecular dynamics simulations revealed that the cytoplasmic torus is stabilized by its core subdomain 3, about which subdomains 2 and 4 hinge, varying the flexible outside cleft implicated in gatekeeper and substrate binding. In light of patterns of surface conservation, deprotonation, and structural motion, the location of previously identified critical residues suggest that gatekeeper binds a cleft buried between neighboring subdomain 4s. Simulations suggest that a local pH change from 5 to 7.2 stabilizes the subdomain 3 hinge and narrows the central aperture of the nonameric torus. Our results are consistent with a model of local pH sensing at SctV, where pH-dependent dynamics of SctV cytoplasmic domain affect binding of gatekeeper complex.
Validation Report
SummaryFull reportAbout validation report
History
DepositionNov 6, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 14, 2021Provider: repository / Type: Initial release
Revision 1.1Apr 21, 2021Group: Database references / Category: citation / Item: _citation.journal_volume

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Assembly

Deposited unit
A: Secretion system apparatus protein SsaV


Theoretical massNumber of molelcules
Total (without water)76,2211
Polymers76,2211
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area18130 Å2

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Components

#1: Protein Secretion system apparatus protein SsaV / Secretion


Mass: 76221.359 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720) (bacteria)
Strain: LT2 / SGSC1412 / ATCC 700720 / Gene: ssaV, STM1414 / Plasmid: pQlinkN / Details (production host): pQlinkN-t5-ssaV6his-ampR / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C41 / 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: Homo-nonameric ring complex of SsaV (SctV) type III secretion system export apparatus protein.Type three secretion system
Type: ORGANELLE OR CELLULAR COMPONENT
Details: Full length protein including transmembrane domain recombinantly expressed in E.coli C41 and extracted from membrane fraction with detergent DDM. Transmembrane domain present but not resolved.
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.68 MDa / Experimental value: YES
Source (natural)Organism: Salmonella enterica (bacteria) / Strain: LT2
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: C41(DE3) / Plasmid: pQlinkN-ssaV6his
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris-HClTris1
250 mMNaClSodium chloride1
30.03 %n-Dodecyl-beta-D-maltoside1
45 mMDithiothreitol1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was monodisperse on a continuous carbon film.
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blot 2.5s

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 12 sec. / Electron dose: 70 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 6729
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 40

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Processing

EM software
IDNameVersionCategory
1Xmippparticle selection
2EPUimage acquisition
4CTFFINDCTF correction
7ISOLDEmodel fitting
8Cootmodel fitting
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
13RELION33D reconstruction
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 988000
Details: interactive training particle picker in Xmipp, used in the Scipion wrapper
SymmetryPoint symmetry: C9 (9 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 361000 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER

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