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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
Sample
  • Organelle or cellular component: Homo-nonameric ring complex of SsaV (SctV) type III secretion system export apparatus protein.Type 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 (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.
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
  • Imaged by UCSF Chimera
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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
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11928.png

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

Histogram

Histogram (log scale)
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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Mask #1

Fileemd_11928_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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
Components
  • Organelle or cellular component: Homo-nonameric ring complex of SsaV (SctV) type III secretion system export apparatus protein.Type three secretion system
    • Protein or peptide: Secretion system apparatus protein SsaVSecretion

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Supramolecule #1: Homo-nonameric ring complex of SsaV (SctV) type III secretion sys...

SupramoleculeName: 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.
Source (natural)Organism: Salmonella enterica (bacteria) / Strain: LT2
Molecular weightExperimental: 680 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: C41(DE3) / Recombinant plasmid: pQlinkN-ssaV6his

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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: 1 / Enantiomer: LEVO
Source (natural)Organism: Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720) (bacteria)
Strain: LT2 / SGSC1412 / ATCC 700720
Molecular weightTheoretical: 76.221359 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MRSWLGEGVR AQQWLSVCAG RQDMVLATVL LIAIVMMLLP LPTWMVDILI TINLMFSVIL LLIAIYLSDP LDLSVFPSLL LITTLYRLS LTISTSRLVL LQHNAGNIVD AFGKFVVGGN LTVGLVVFTI ITIVQFIVIT KGIERVAEVS ARFSLDGMPG K QMSIDGDL ...String:
MRSWLGEGVR AQQWLSVCAG RQDMVLATVL LIAIVMMLLP LPTWMVDILI TINLMFSVIL LLIAIYLSDP LDLSVFPSLL LITTLYRLS LTISTSRLVL LQHNAGNIVD AFGKFVVGGN LTVGLVVFTI ITIVQFIVIT KGIERVAEVS ARFSLDGMPG K QMSIDGDL RAGVIDADHA RTLRQHVQQE SRFLGAMDGA MKFVKGDTIA GIIVVLVNII GGIIIAIVQY DMSMSEAVHT YS VLSIGDG LCGQIPSLLI SLSAGIIVTR VPGEKRQNLA TELSSQIARQ PQSLILTAVV LMLLALIPGF PFITLAFFSA LLA LPIILI RRKKSVVSAN GVEAPEKDSM VPGACPLILR LSPTLHSADL IRDIDAMRWF LFEDTGVPLP EVNIEVLPEP TEKL TVLLY QEPVFSLSIP AQADYLLIGA DASVVGDSQT LPNGMGQICW LTKDMAHKAQ GFGLDVFAGS QRISALLKCV LLRHM GEFI GVQETRYLMN AMEKNYSELV KELQRQLPIN KIAETLQRLV SERVSIRDLR LIFGTLIDWA PREKDVLMLT EYVRIA LRR HILRRLNPEG KPLPILRIGE GIENLVRESI RQTAMGTYTA LSSRHKTQIL QLIEQALKQS AKLFIVTSVD TRRFLRK IT EATLFDVPIL SWQELGEESL IQVVESIDLS EEELADNEEH HHHHH

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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

Concentration1 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
50.0 mMTris-HClTris
50.0 mMNaClSodium chloride
0.03 %n-Dodecyl-beta-D-maltoside
5.0 mMDithiothreitol
GridModel: 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: 0.30000000000000004 nm / Support film - #1 - Film type ID: 2 / Support film - #1 - Material: CARBON / Support film - #1 - topology: HOLEY / Support film - #1 - Film thickness: 12.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blot 2.5s.
DetailsSample was monodisperse on a continuous carbon film.

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm 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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 988000
Details: interactive training particle picker in Xmipp, used in the Scipion wrapper
CTF correctionSoftware - Name: CTFFIND
Startup modelType 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.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied 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
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: OTHER
Output model

PDB-7awa:
SctV (SsaV) cytoplasmic domain

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