+Open data
-Basic information
Entry | Database: PDB / ID: 7osl | ||||||
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Title | Cryo-EM structure of nonameric EPEC SctV-C | ||||||
Components | Translocator EscV | ||||||
Keywords | PROTEIN TRANSPORT / T3SS / SctV nonamer / export gate | ||||||
Function / homology | Function and homology information | ||||||
Biological species | Escherichia coli O127:H6 (bacteria) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å | ||||||
Authors | Fahrenkamp, D. / Wald, J. / Yuan, B. / Marlovits, T.C. | ||||||
Funding support | Austria, 1items
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Citation | Journal: J Mol Biol / Year: 2021 Title: Structural Dynamics of the Functional Nonameric Type III Translocase Export Gate. Authors: Biao Yuan / Athina G Portaliou / Rinky Parakra / Jochem H Smit / Jiri Wald / Yichen Li / Bindu Srinivasu / Maria S Loos / Harveer Singh Dhupar / Dirk Fahrenkamp / Charalampos G Kalodimos / ...Authors: Biao Yuan / Athina G Portaliou / Rinky Parakra / Jochem H Smit / Jiri Wald / Yichen Li / Bindu Srinivasu / Maria S Loos / Harveer Singh Dhupar / Dirk Fahrenkamp / Charalampos G Kalodimos / Franck Duong van Hoa / Thorben Cordes / Spyridoula Karamanou / Thomas C Marlovits / Anastassios Economou / Abstract: Type III protein secretion is widespread in Gram-negative pathogens. It comprises the injectisome with a surface-exposed needle and an inner membrane translocase. The translocase contains the SctRSTU ...Type III protein secretion is widespread in Gram-negative pathogens. It comprises the injectisome with a surface-exposed needle and an inner membrane translocase. The translocase contains the SctRSTU export channel enveloped by the export gate subunit SctV that binds chaperone/exported clients and forms a putative ante-chamber. We probed the assembly, function, structure and dynamics of SctV from enteropathogenic E. coli (EPEC). In both EPEC and E. coli lab strains, SctV forms peripheral oligomeric clusters that are detergent-extracted as homo-nonamers. Membrane-embedded SctV is necessary and sufficient to act as a receptor for different chaperone/exported protein pairs with distinct C-domain binding sites that are essential for secretion. Negative staining electron microscopy revealed that peptidisc-reconstituted His-SctV forms a tripartite particle of ∼22 nm with a N-terminal domain connected by a short linker to a C-domain ring structure with a ∼5 nm-wide inner opening. The isolated C-domain ring was resolved with cryo-EM at 3.1 Å and structurally compared to other SctV homologues. Its four sub-domains undergo a three-stage "pinching" motion. Hydrogen-deuterium exchange mass spectrometry revealed this to involve dynamic and rigid hinges and a hyper-flexible sub-domain that flips out of the ring periphery and binds chaperones on and between adjacent protomers. These motions are coincident with local conformational changes at the pore surface and ring entry mouth that may also be modulated by the ATPase inner stalk. We propose that the intrinsic dynamics of the SctV protomer are modulated by chaperones and the ATPase and could affect allosterically the other subunits of the nonameric ring during secretion. | ||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7osl.cif.gz | 964.5 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7osl.ent.gz | 820.4 KB | Display | PDB format |
PDBx/mmJSON format | 7osl.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7osl_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 7osl_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | 7osl_validation.xml.gz | 78.1 KB | Display | |
Data in CIF | 7osl_validation.cif.gz | 118 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/os/7osl ftp://data.pdbj.org/pub/pdb/validation_reports/os/7osl | HTTPS FTP |
-Related structure data
Related structure data | 13054MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | Mass: 37952.457 Da / Num. of mol.: 9 / Mutation: no Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli O127:H6 (strain E2348/69 / EPEC) (bacteria) Strain: E2348/69 / EPEC / Gene: escV, E2348C_3949 Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) References: UniProt: B7UMA7 |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: EPEC SctV-C nonamer / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT | |||||||||||||||
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Molecular weight | Value: 379.2 kDa/nm / Experimental value: YES | |||||||||||||||
Source (natural) | Organism: Escherichia coli O127:H6 str. E2348/69 (bacteria) | |||||||||||||||
Source (recombinant) | Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) | |||||||||||||||
Buffer solution | pH: 8 | |||||||||||||||
Buffer component |
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Specimen | Conc.: 1.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: The sample was mono-disperse. | |||||||||||||||
Specimen support | Grid material: GOLD / Grid type: Quantifoil R1.2/1.3 | |||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Cs: 2.7 mm / C2 aperture diameter: 70 µm |
Image recording | Electron dose: 41.25 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 3739 |
-Processing
EM software |
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CTF correction | Type: NONE | |||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 660798 | |||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 105670 / Symmetry type: POINT |