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- EMDB-11928: SctV (SsaV) cytoplasmic domain -

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Basic information

Entry
Database: EMDB / ID: EMD-11928
TitleSctV (SsaV) cytoplasmic domain
Map data
SampleHomo-nonameric ring complex of SsaV (SctV) type III secretion system export apparatus protein.Type three secretion system
  • Secretion system apparatus protein SsaVSecretion
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 enterica (bacteria) / Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsMatthews-Palmer TRS / Gonzalez-Rodriguez N / Calcraft T / Lagercrantz S / Zachs T / Yu XJ / Grabe G / Holden D / Nans A / Rosenthal P ...Matthews-Palmer TRS / Gonzalez-Rodriguez N / Calcraft T / Lagercrantz S / Zachs T / Yu XJ / Grabe G / Holden D / Nans A / Rosenthal P / Rouse S / Beeby M
Funding support United Kingdom, 4 items
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 ReportSummary, Full report, XML, About validation report
History
DepositionNov 6, 2020-
Header (metadata) releaseApr 14, 2021-
Map releaseApr 14, 2021-
UpdateApr 21, 2021-
Current statusApr 21, 2021Processing 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-7awa
  • Surface level: 0.013
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7awa
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Structure viewerEM map:
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Supplemental images

Downloads & links

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Map

FileDownload / File: emd_11928.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 300 pix.
= 324. Å
1.08 Å/pix.
x 300 pix.
= 324. Å
1.08 Å/pix.
x 300 pix.
= 324. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.013 / Movie #1: 0.013
Minimum - Maximum-0.032984994 - 0.07650644
Average (Standard dev.)0.0002004739 (±0.0020256157)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 324.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z324.000324.000324.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0330.0770.000

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Supplemental data

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Segmentation: #1

Fileemd_11928_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire Homo-nonameric ring complex of SsaV (SctV) type III secretion sys...

EntireName: Homo-nonameric ring complex of SsaV (SctV) type III secretion system export apparatus protein.Type three secretion system
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.
Number of components: 2

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Component #1: cellular-component, Homo-nonameric ring complex of SsaV (SctV) ty...

Cellular-componentName: Homo-nonameric ring complex of SsaV (SctV) type III secretion system export apparatus protein.Type three secretion system
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.
Recombinant expression: No
MassExperimental: 680 kDa
SourceSpecies: Salmonella enterica (bacteria) / Strain: LT2
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pQlinkN-ssaV6his / Strain: C41(DE3)

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Component #2: protein, Secretion system apparatus protein SsaV

ProteinName: Secretion system apparatus protein SsaVSecretion / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 76.221359 kDa
SourceSpecies: Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720) (bacteria)
Strain: LT2 / SGSC1412 / ATCC 700720
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 1 mg/mL / pH: 8
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: OTHER / Temperature: 277 K / Humidity: 100 % / Details: blot 2.5s.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 70 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD / Energy filter: GIF Bioquantum
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 6729

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C9 (9 fold cyclic) / Number of projections: 361000
3D reconstructionSoftware: RELION / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Output model

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