|Entry||Database: EMDB / ID: EMD-22727|
|Title||EsxE-EsxF pentameric pore|
|Sample||Hetero-pentameric pore complex formed from EsxE/EsxF heterodimers at pH 6.5.:|
|Biological species||Mycobacterium tuberculosis (bacteria) / Mycobacterium tuberculosis H37RV (bacteria)|
|Method||single particle reconstruction / negative staining / Resolution: 17.6 Å|
|Authors||Tak U / Dokland T / Niederweis M|
|Funding support|| United States, 1 items |
|Citation||Journal: Nat Commun / Year: 2021|
Title: Pore-forming Esx proteins mediate toxin secretion by Mycobacterium tuberculosis.
Authors: Uday Tak / Terje Dokland / Michael Niederweis /
Abstract: Mycobacterium tuberculosis secretes the tuberculosis necrotizing toxin (TNT) to kill host cells. Here, we show that the WXG100 proteins EsxE and EsxF are essential for TNT secretion. EsxE and EsxF ...Mycobacterium tuberculosis secretes the tuberculosis necrotizing toxin (TNT) to kill host cells. Here, we show that the WXG100 proteins EsxE and EsxF are essential for TNT secretion. EsxE and EsxF form a water-soluble heterodimer (EsxEF) that assembles into oligomers and long filaments, binds to membranes, and forms stable membrane-spanning channels. Electron microscopy of EsxEF reveals mainly pentameric structures with a central pore. Mutations of both WXG motifs and of a GXW motif do not affect dimerization, but abolish pore formation, membrane deformation and TNT secretion. The WXG/GXW mutants are locked in conformations with altered thermostability and solvent exposure, indicating that the WXG/GXW motifs are molecular switches controlling membrane interaction and pore formation. EsxF is accessible on the bacterial cell surface, suggesting that EsxEF form an outer membrane channel for toxin export. Thus, our study reveals a protein secretion mechanism in bacteria that relies on pore formation by small WXG proteins.
|Validation Report||Summary, Full report, XML, About validation report|
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_22727.map.gz / Format: CCP4 / Size: 12.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.23 Å|
|Symmetry||Space group: 1|
CCP4 map header:
|Projections & Slices|
|Projections & Slices|
-Half map: Half map even
|Annotation||Half map even|
|Projections & Slices|
-Half map: Half map odd
-Entire Hetero-pentameric pore complex formed from EsxE/EsxF heterodimers...
|Entire||Name: Hetero-pentameric pore complex formed from EsxE/EsxF heterodimers at pH 6.5.|
Number of components: 2
-Component #1: protein, Hetero-pentameric pore complex formed from EsxE/EsxF het...
|Protein||Name: Hetero-pentameric pore complex formed from EsxE/EsxF heterodimers at pH 6.5.|
Recombinant expression: No
|Source||Species: Mycobacterium tuberculosis (bacteria) / Strain: H37Rv|
|Source (engineered)||Expression System: Escherichia coli BL21(DE3) (bacteria) / Vector: pML3924 / Strain: BL21|
|Source (natural)||Location in cell: Membrane, Periplasm, Secreted|
-Component #2: nucleic-acid, EsxE/EsxF Pentamer
|nucleic acid||Name: EsxE/EsxF Pentamer / Class: OTHER|
Details: Hetero-Pentameric pore formed from hetero-oligomers of EsxE/EsxF.
Structure: OTHER / Synthetic: No
Sequence: MGADDTLRVE PAVMQGFAAS LDGAAEHLAV QLAELDAQVG QMLGGWRGAS GSAYGSAWEL WHRGAGEVQL GLSMLAAAIA HAGAGYQHNE TASAQVLREV GGGVDPTVLA DAVARMAEFG RHVEELVAEI ESLVTRLHVT WTGEGAAAHA EAQRHWAAGE AMMRQALAQL ...Sequence:
MGADDTLRVE PAVMQGFAAS LDGAAEHLAV QLAELDAQVG QMLGGWRGAS GSAYGSAWEL WHRGAGEVQL GLSMLAAAIA HAGAGYQHNE TASAQVLREV GGGVDPTVLA DAVARMAEFG RHVEELVAEI ESLVTRLHVT WTGEGAAAHA EAQRHWAAGE AMMRQALAQL TAAGQSAHAN YTGAMATNLG MWS
|Source||Species: Mycobacterium tuberculosis H37RV (bacteria)|
|Specimen||Specimen state: Particle / Method: negative staining|
|Sample solution||Specimen conc.: 0.008 mg/mL|
Buffer solution: Solutions were made fresh, autoclaved, and filter sterilized to prevent microbial contamination. 25 mM sodium phosophate, 150 mM NaCl pH 6.5
|Staining||3 microliters of sample was placed on glow discharged grids for 1 minute, then blotted with filter paper, washed / blotted 2x with milli-q water, then stained with 1% uranyl formate for 10 seconds, blotted, followed by staining with 1% uranyl formate for 1 minute. The stain was then blotted and the grid was allowed to air dry.|
|Vitrification||Cryogen name: NONE|
-Electron microscopy imaging
Model: Tecnai F20 / Image courtesy: FEI Company
|Imaging||Microscope: FEI TECNAI F20|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 30 e/Å2 / Illumination mode: SPOT SCAN|
|Lens||Imaging mode: BRIGHT FIELD|
|Specimen Holder||Model: OTHER|
|Image acquisition||Number of digital images: 52|
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