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- EMDB-22727: EsxE-EsxF pentameric pore -

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Basic information

Entry
Database: EMDB / ID: EMD-22727
TitleEsxE-EsxF pentameric pore
Map dataEsxE/EsxF (Mycobacterium tuberculosis) hetero-pentameric pore produced recombinantly in E.coli. Single particle reconstruction in 1% uranyl formate. Reconstruction performed with EMAN2.2.
Sample
  • Complex: Hetero-pentameric pore complex formed from EsxE/EsxF heterodimers at pH 6.5.
    • Other: EsxE/EsxF Pentamer
Biological speciesMycobacterium tuberculosis (bacteria) / Mycobacterium tuberculosis H37RV (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 17.6 Å
AuthorsTak U / Dokland T / Niederweis M
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI121354 United States
CitationJournal: 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.
History
DepositionSep 26, 2020-
Header (metadata) releaseJan 20, 2021-
Map releaseJan 20, 2021-
UpdateJan 27, 2021-
Current statusJan 27, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.66
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.66
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22727.png

Downloads & links

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Map

FileDownload / File: emd_22727.map.gz / Format: CCP4 / Size: 12.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEsxE/EsxF (Mycobacterium tuberculosis) hetero-pentameric pore produced recombinantly in E.coli. Single particle reconstruction in 1% uranyl formate. Reconstruction performed with EMAN2.2.
Voxel sizeX=Y=Z: 1.23 Å
Density
Contour LevelBy AUTHOR: 0.66 / Movie #1: 0.66
Minimum - Maximum-2.7337554 - 3.1344934
Average (Standard dev.)0.01068672 (±0.32392514)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-75-75-75
Dimensions150150150
Spacing150150150
CellA=B=C: 184.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.231.231.23
M x/y/z150150150
origin x/y/z0.0000.0000.000
length x/y/z184.500184.500184.500
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS-75-75-75
NC/NR/NS150150150
D min/max/mean-2.7343.1340.011

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Supplemental data

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Mask #1

Fileemd_22727_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Mask #2

Fileemd_22727_msk_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map even

Fileemd_22727_half_map_1.map
AnnotationHalf map even
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map odd

Fileemd_22727_half_map_2.map
AnnotationHalf map odd
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Hetero-pentameric pore complex formed from EsxE/EsxF heterodimers...

EntireName: Hetero-pentameric pore complex formed from EsxE/EsxF heterodimers at pH 6.5.
Components
  • Complex: Hetero-pentameric pore complex formed from EsxE/EsxF heterodimers at pH 6.5.
    • Other: EsxE/EsxF Pentamer

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Supramolecule #1: Hetero-pentameric pore complex formed from EsxE/EsxF heterodimers...

SupramoleculeName: Hetero-pentameric pore complex formed from EsxE/EsxF heterodimers at pH 6.5.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mycobacterium tuberculosis (bacteria) / Strain: H37Rv / Location in cell: Membrane, Periplasm, Secreted
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21 / Recombinant plasmid: pML3924

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Macromolecule #1: EsxE/EsxF Pentamer

MacromoleculeName: EsxE/EsxF Pentamer / type: other / ID: 1
Details: Hetero-Pentameric pore formed from hetero-oligomers of EsxE/EsxF.
Classification: other
Source (natural)Organism: Mycobacterium tuberculosis H37RV (bacteria)
SequenceString: MGADDTLRVE PAVMQGFAAS LDGAAEHLAV QLAELDAQVG QMLGGWRGAS GSAYGSAWEL WHRGAGEVQL GLSMLAAAIA HAGAGYQHNE TASAQVLREV GGG VDPTVL ADAVARMAEF GRHVEELVAE IESLVTRLHV TWTGEGAAAH AEAQRHWAAG EAMMRQALAQ ...String:
MGADDTLRVE PAVMQGFAAS LDGAAEHLAV QLAELDAQVG QMLGGWRGAS GSAYGSAWEL WHRGAGEVQL GLSMLAAAIA HAGAGYQHNE TASAQVLREV GGG VDPTVL ADAVARMAEF GRHVEELVAE IESLVTRLHV TWTGEGAAAH AEAQRHWAAG EAMMRQALAQ LTAAGQSAHA NYTGAMATNL GMWS
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.008 mg/mL
BufferpH: 6.5 / Component - Formula: NaClSodium chloride / Component - Name: sodium phosphate
Details: Solutions were made fresh, autoclaved, and filter sterilized to prevent microbial contamination. 25 mM sodium phosophate, 150 mM NaCl pH 6.5
StainingType: NEGATIVE / Material: Uranyl Formate
Details: 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 ...Details: 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.
DetailsThis sample was well dispersed.

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 52 / Average exposure time: 1.0 sec. / Average electron dose: 30.0 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 19179
CTF correctionDetails: CTF was applied after particle selection and was set for low resolution (CTF Autoprocessing).
Startup modelType of model: NONE
Details: C5 symmetry was applied due to the clear pentameric symmetry found in reference-free 2D class averages.
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionNumber classes used: 50 / Applied symmetry - Point group: C5 (5 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 17.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: EMAN (ver. 2.2) / Number images used: 19179
FSC plot (resolution estimation)

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