+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-13054 | |||||||||
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Title | Cryo-EM structure of nonameric EPEC SctV-C | |||||||||
Map data | Post-processed map_masked | |||||||||
Sample |
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Keywords | T3SS / SctV nonamer / export gate / PROTEIN TRANSPORT | |||||||||
Function / homology | Function and homology information | |||||||||
Biological species | Escherichia coli O127:H6 str. E2348/69 (bacteria) / Escherichia coli O127:H6 (strain E2348/69 / EPEC) (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.1 Å | |||||||||
Authors | Yuan B / Wald J / Fahrenkamp D / Marlovits TC | |||||||||
Funding support | Austria, 1 items
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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 | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_13054.map.gz | 7.4 MB | EMDB map data format | |
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Header (meta data) | emd-13054-v30.xml emd-13054.xml | 17.3 KB 17.3 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_13054_fsc.xml | 8.5 KB | Display | FSC data file |
Images | emd_13054.png | 139.8 KB | ||
Masks | emd_13054_msk_1.map | 52.7 MB | Mask map | |
Filedesc metadata | emd-13054.cif.gz | 5.7 KB | ||
Others | emd_13054_half_map_1.map.gz emd_13054_half_map_2.map.gz | 40.7 MB 40.7 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-13054 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-13054 | HTTPS FTP |
-Validation report
Summary document | emd_13054_validation.pdf.gz | 740.4 KB | Display | EMDB validaton report |
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Full document | emd_13054_full_validation.pdf.gz | 740 KB | Display | |
Data in XML | emd_13054_validation.xml.gz | 14 KB | Display | |
Data in CIF | emd_13054_validation.cif.gz | 20 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-13054 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-13054 | HTTPS FTP |
-Related structure data
Related structure data | 7oslMC 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_13054.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Post-processed map_masked | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.1 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_13054_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: half-map1 from auto-refinement
File | emd_13054_half_map_1.map | ||||||||||||
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Annotation | half-map1 from auto-refinement | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half-map 2 from autorefinement
File | emd_13054_half_map_2.map | ||||||||||||
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Annotation | Half-map 2 from autorefinement | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : EPEC SctV-C nonamer
Entire | Name: EPEC SctV-C nonamer |
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Components |
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-Supramolecule #1: EPEC SctV-C nonamer
Supramolecule | Name: EPEC SctV-C nonamer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: Escherichia coli O127:H6 str. E2348/69 (bacteria) |
Molecular weight | Theoretical: 379.2 kDa/nm |
-Macromolecule #1: Translocator EscV
Macromolecule | Name: Translocator EscV / type: protein_or_peptide / ID: 1 / Number of copies: 9 / Enantiomer: LEVO |
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Source (natural) | Organism: Escherichia coli O127:H6 (strain E2348/69 / EPEC) (bacteria) Strain: E2348/69 / EPEC |
Molecular weight | Theoretical: 37.952457 KDa |
Recombinant expression | Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) |
Sequence | String: NISPGAEPLI LNLSSNIYSS DITQQIEVMR WNFFEESGIP LPKIIVNPVK NNDSAIEFLL YQESIYKDTL IDDTVYFEAG HAEISFEFV QEKLSTNSIV YKTNKTNQQL AHLTGMDVYA TTNDKITFLL KKLVLSNAKE FIGVQETRYL MDIMERKYNE L VKELQRQL ...String: NISPGAEPLI LNLSSNIYSS DITQQIEVMR WNFFEESGIP LPKIIVNPVK NNDSAIEFLL YQESIYKDTL IDDTVYFEAG HAEISFEFV QEKLSTNSIV YKTNKTNQQL AHLTGMDVYA TTNDKITFLL KKLVLSNAKE FIGVQETRYL MDIMERKYNE L VKELQRQL GLSKIVDILQ RLVEENVSIR DLRTIFETLI FWSTKEKDVV ILCEYVRIAL RRHILGRYSV SGTLLNVWLI GS DIENELR ESIRQTSSGS YLNISPERTE QIIGFLKNIM NPTGNGVILT ALDIRRYVKK MIEGSFPSVP VLSFQEVGNN IEL KVLGTV NDFRA UniProtKB: Translocator EscV |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1.8 mg/mL | |||||||||
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Buffer | pH: 8 Component:
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Grid | Model: Quantifoil R1.2/1.3 / Material: GOLD / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR | |||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV | |||||||||
Details | The sample was mono-disperse. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 3739 / Average electron dose: 41.25 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |