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Yorodumi- EMDB-0135: Electron cryo-microscopy of the Type VI secretion "pre-firing" Vg... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-0135 | |||||||||
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Title | Electron cryo-microscopy of the Type VI secretion "pre-firing" VgrG1-Tse6-EagT6-EF-Tu-Tsi6 complex | |||||||||
Map data | The "pre-firing" VgrG1-Tse6-EagT6-EF-Tu-Tsi6 complex (PFC) | |||||||||
Sample |
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Keywords | Bacterial Type VI effector complex / T6SS chaperone-effector complex / Tse6-loaded VgrG1 complex / NAD(P)+ Glycohydrolase / TOXIN | |||||||||
Function / homology | Function and homology information type VI protein secretion system complex / protein secretion by the type VI secretion system / extracellular region Similarity search - Function | |||||||||
Biological species | Pseudomonas aeruginosa PAO1 (bacteria) / Escherichia coli BL21 (bacteria) / Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.2 Å | |||||||||
Authors | Quentin D / Raunser S | |||||||||
Funding support | Germany, 1 items
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Citation | Journal: Nat Microbiol / Year: 2018 Title: Mechanism of loading and translocation of type VI secretion system effector Tse6. Authors: Dennis Quentin / Shehryar Ahmad / Premy Shanthamoorthy / Joseph D Mougous / John C Whitney / Stefan Raunser / Abstract: The type VI secretion system (T6SS) primarily functions to mediate antagonistic interactions between contacting bacterial cells, but also mediates interactions with eukaryotic hosts. This molecular ...The type VI secretion system (T6SS) primarily functions to mediate antagonistic interactions between contacting bacterial cells, but also mediates interactions with eukaryotic hosts. This molecular machine secretes antibacterial effector proteins by undergoing cycles of extension and contraction; however, how effectors are loaded into the T6SS and subsequently delivered to target bacteria remains poorly understood. Here, using electron cryomicroscopy, we analysed the structures of the Pseudomonas aeruginosa effector Tse6 loaded onto the T6SS spike protein VgrG1 in solution and embedded in lipid nanodiscs. In the absence of membranes, Tse6 stability requires the chaperone EagT6, two dimers of which interact with the hydrophobic transmembrane domains of Tse6. EagT6 is not directly involved in Tse6 delivery but is crucial for its loading onto VgrG1. VgrG1-loaded Tse6 spontaneously enters membranes and its toxin domain translocates across a lipid bilayer, indicating that effector delivery by the T6SS does not require puncturing of the target cell inner membrane by VgrG1. Eag chaperone family members from diverse Proteobacteria are often encoded adjacent to putative toxins with predicted transmembrane domains and we therefore anticipate that our findings will be generalizable to numerous T6SS-exported membrane-associated effectors. | |||||||||
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_0135.map.gz | 7.5 MB | EMDB map data format | |
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Header (meta data) | emd-0135-v30.xml emd-0135.xml | 14.8 KB 14.8 KB | Display Display | EMDB header |
Images | emd_0135.png | 57.2 KB | ||
Filedesc metadata | emd-0135.cif.gz | 6.4 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-0135 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-0135 | HTTPS FTP |
-Validation report
Summary document | emd_0135_validation.pdf.gz | 203 KB | Display | EMDB validaton report |
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Full document | emd_0135_full_validation.pdf.gz | 202.1 KB | Display | |
Data in XML | emd_0135_validation.xml.gz | 6.7 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-0135 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-0135 | HTTPS FTP |
-Related structure data
Related structure data | 6h3lMC 0136C 6h3nC 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_0135.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | The "pre-firing" VgrG1-Tse6-EagT6-EF-Tu-Tsi6 complex (PFC) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.14 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Type VI secretion "pre-firing" VgrG1-Tse6-EagT6-EF-Tu-Tsi6 complex
Entire | Name: Type VI secretion "pre-firing" VgrG1-Tse6-EagT6-EF-Tu-Tsi6 complex |
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Components |
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-Supramolecule #1: Type VI secretion "pre-firing" VgrG1-Tse6-EagT6-EF-Tu-Tsi6 complex
Supramolecule | Name: Type VI secretion "pre-firing" VgrG1-Tse6-EagT6-EF-Tu-Tsi6 complex type: complex / ID: 1 / Parent: 0 / Macromolecule list: all Details: The type VI "pre-firing" complex consists of a single copy of Tse6, EF-Tu, Tsi6 and trimeric VgrG1 as well as two copies of dimeric EagT6. |
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Molecular weight | Theoretical: 43 KDa |
-Supramolecule #2: Valine-glycine repeat protein 1
Supramolecule | Name: Valine-glycine repeat protein 1 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: all / Details: VgrG1 forms a trimer - UniProt Identifier: Q9I741 |
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Source (natural) | Organism: Pseudomonas aeruginosa PAO1 (bacteria) |
-Supramolecule #3: Type VI secretion exported 6
Supramolecule | Name: Type VI secretion exported 6 / type: complex / ID: 3 / Parent: 1 / Details: Tse6 - UniProt identifier: Q9I739 |
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Source (natural) | Organism: Pseudomonas aeruginosa PAO1 (bacteria) |
-Supramolecule #4: Type VI secretion immunity 6
Supramolecule | Name: Type VI secretion immunity 6 / type: complex / ID: 4 / Parent: 1 / Details: Tsi6 - UniProt identifier: Q9I740 |
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Source (natural) | Organism: Pseudomonas aeruginosa PAO1 (bacteria) |
-Supramolecule #5: Effector associated gene with tse6
Supramolecule | Name: Effector associated gene with tse6 / type: complex / ID: 5 / Parent: 1 Details: EagT6 forms dimers - UniProt identifier: Q9I738. Two dimers are present in the EM map. |
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Source (natural) | Organism: Pseudomonas aeruginosa PAO1 (bacteria) |
-Supramolecule #6: Elongation factor Tu 1
Supramolecule | Name: Elongation factor Tu 1 / type: complex / ID: 6 / Parent: 1 / Details: EF-Tu - UniProt identifier: P0CE47 |
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Source (natural) | Organism: Escherichia coli BL21 (bacteria) |
-Macromolecule #1: VgrG1
Macromolecule | Name: VgrG1 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) (bacteria) |
Molecular weight | Theoretical: 72.098375 KDa |
Recombinant expression | Organism: Escherichia coli BL21 (bacteria) |
Sequence | String: MQLTRLVQVD CPLGPDVLLL QRMEGREELG RLFAYELHLV SENPNLPLEQ LLGKPMSLSL ELPGGSRRFF HGIVARCSQV AGHGQFAGY QATLRPWPWL LTRTSDCRIF QNQSVPEIIK QVFRNLGFSD FEDALTRPYR EWEYCVQYRE TSFDFISRLM E QEGIYYWF ...String: MQLTRLVQVD CPLGPDVLLL QRMEGREELG RLFAYELHLV SENPNLPLEQ LLGKPMSLSL ELPGGSRRFF HGIVARCSQV AGHGQFAGY QATLRPWPWL LTRTSDCRIF QNQSVPEIIK QVFRNLGFSD FEDALTRPYR EWEYCVQYRE TSFDFISRLM E QEGIYYWF RHEQKRHILV LSDAYGAHRS PGGYASVPYY PPTLGHRERD HFFDWQMARE VQPGSLTLND YDFQRPGARL EV RSNIARP HAAADYPLYD YPGEYVQSQD GEQYARNRIE AIQAQHERVR LRGVVRGIGA GHLFRLSGYP RDDQNREYLV VGA EYRVVQ ELYETGSGGA GSQFESELDC IDASQSFRLL PQTPVPVVRG PQTAVVVGPK GEEIWTDQYG RVKVHFHWDR HDQS NENSS CWIRVSQAWA GKNWGSMQIP RIGQEVIVSF LEGDPDRPII TGRVYNAEQT VPYELPANAT QSGMKSRSSK GGTPA NFNE IRMEDKKGAE QLYIHAERNQ DNLVENDASL SVGHDRNKSI GHDELARIGN NRTRAVKLND TLLVGGAKSD SVTGTY LIE AGAQIRLVCG KSVVEFNADG TINISGSAFN LYASGNGNID TGGRLDLNSG GASEVDAKGK GVQGTIDGQV QAMFPPP AK G UniProtKB: Type VI secretion system spike protein VgrG1a |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.015 mg/mL | |||||||||
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Buffer | pH: 8 Component:
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Grid | Model: Quantifoil R2/1 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2 / Pretreatment - Type: GLOW DISCHARGE | |||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 298 K / Instrument: GATAN CRYOPLUNGE 3 |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Spherical aberration corrector: Cs corrected microscope |
Image recording | Film or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Number real images: 11642 / Average exposure time: 1.5 sec. / Average electron dose: 60.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 0.01 mm / Nominal defocus max: 4.2 µm / Nominal defocus min: 1.7 µm / Nominal magnification: 59000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Startup model | Type of model: EMDB MAP EMDB ID: |
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Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: SPHIRE / Software - details: meridien / Number images used: 55000 |
Initial angle assignment | Type: MAXIMUM LIKELIHOOD |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |