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
Journal: 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.
History
Deposition
Nov 6, 2020
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Header (metadata) release
Apr 14, 2021
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Map release
Apr 14, 2021
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Update
May 1, 2024
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Current status
May 1, 2024
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Entire : Homo-nonameric ring complex of SsaV (SctV) type III secretion sys...
Entire
Name: Homo-nonameric ring complex of SsaV (SctV) type III secretion system export apparatus protein.
Components
Organelle or cellular component: Homo-nonameric ring complex of SsaV (SctV) type III secretion system export apparatus protein.
Protein or peptide: Secretion system apparatus protein SsaV
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Supramolecule #1: Homo-nonameric ring complex of SsaV (SctV) type III secretion sys...
Supramolecule
Name: Homo-nonameric ring complex of SsaV (SctV) type III secretion system export apparatus protein. type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all 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.
UniProtKB: Secretion system apparatus protein SsaV
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Concentration
1 mg/mL
Buffer
pH: 8 Component:
Concentration
Name
Formula
50.0 mM
Tris-HCl
50.0 mM
NaCl
0.03 %
n-Dodecyl-beta-D-maltoside
5.0 mM
Dithiothreitol
Grid
Model: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - #0 - Film type ID: 1 / Support film - #0 - Material: CARBON / Support film - #0 - topology: CONTINUOUS / Support film - #0 - Film thickness: 0.3 / Support film - #1 - Film type ID: 2 / Support film - #1 - Material: CARBON / Support film - #1 - topology: HOLEY / Support film - #1 - Film thickness: 12 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
Vitrification
Cryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blot 2.5s.
Details
Sample was monodisperse on a continuous carbon film.
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Electron microscopy
Microscope
TFS KRIOS
Specialist optics
Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 2 / Number real images: 6729 / Average exposure time: 12.0 sec. / Average electron dose: 70.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stage
Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
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Image processing
Particle selection
Number selected: 988000 Details: interactive training particle picker in Xmipp, used in the Scipion wrapper
Startup model
Type of model: OTHER Details: Ab initio 3D model generation from a balanced set of 2D views using stochastic gradient descent implemented in Relion. Initial model heavily low-pass filtered.
Final reconstruction
Applied symmetry - Point group: C9 (9 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 361000
Initial angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION
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