+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-11928 | |||||||||||||||
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Title | SctV (SsaV) cytoplasmic domain | |||||||||||||||
Map data | ||||||||||||||||
Sample |
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Function / homology | Function and homology information | |||||||||||||||
Biological species | Salmonella enterica (bacteria) / Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720) (bacteria) | |||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.5 Å | |||||||||||||||
Authors | Matthews-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
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Citation | 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_11928.map.gz | 6.9 MB | EMDB map data format | |
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Header (meta data) | emd-11928-v30.xml emd-11928.xml | 16.6 KB 16.6 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_11928_fsc.xml | 10.7 KB | Display | FSC data file |
Images | emd_11928.png | 130 KB | ||
Masks | emd_11928_msk_1.map | 103 MB | Mask map | |
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-11928 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-11928 | HTTPS FTP |
-Related structure data
Related structure data | 7awaMC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_11928.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Voxel size | X=Y=Z: 1.08 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Mask #1
File | emd_11928_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-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.Type three secretion system |
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Components |
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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. |
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Source (natural) | Organism: Salmonella enterica (bacteria) / Strain: LT2 |
Molecular weight | Experimental: 680 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: C41(DE3) / Recombinant plasmid: pQlinkN-ssaV6his |
-Macromolecule #1: Secretion system apparatus protein SsaV
Macromolecule | Name: Secretion system apparatus protein SsaV / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Salmonella typhimurium (strain LT2 / SGSC1412 / ATCC 700720) (bacteria) Strain: LT2 / SGSC1412 / ATCC 700720 |
Molecular weight | Theoretical: 76.221359 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: 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 |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1 mg/mL | |||||||||||||||
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Buffer | pH: 8 Component:
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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: 0.30000000000000004 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 | |||||||||||||||
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. |
-Electron microscopy
Microscope | TFS KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy |
Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Sample stage | Cooling holder cryogen: NITROGEN |
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 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
-Atomic model buiding 1
Refinement | Protocol: OTHER |
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Output model | PDB-7awa: |