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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 data
SampleHetero-pentameric pore complex formed from EsxE/EsxF heterodimers at pH 6.5.:
nucleic-acidNucleic acid
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.
Validation ReportSummary, Full report, XML, About validation report
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

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)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.23 Å/pix.
x 150 pix.
= 184.5 Å
1.23 Å/pix.
x 150 pix.
= 184.5 Å
1.23 Å/pix.
x 150 pix.
= 184.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

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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Segmentation: #1

Fileemd_22727_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Segmentation: #2

Fileemd_22727_msk_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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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

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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

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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.
Number of components: 2

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Component #1: protein, Hetero-pentameric pore complex formed from EsxE/EsxF het...

ProteinName: Hetero-pentameric pore complex formed from EsxE/EsxF heterodimers at pH 6.5.
Recombinant expression: No
SourceSpecies: 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

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Component #2: nucleic-acid, EsxE/EsxF Pentamer

nucleic acidName: 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
SourceSpecies: Mycobacterium tuberculosis H37RV (bacteria)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: negative staining
Sample solutionSpecimen 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
pH: 6.5
Staining3 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.
VitrificationCryogen name: NONE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 30 e/Å2 / Illumination mode: SPOT SCAN
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: OTHER

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Image acquisition

Image acquisitionNumber of digital images: 52

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C5 (5 fold cyclic) / Number of projections: 19179
3D reconstructionSoftware: EMAN
CTF correction: CTF was applied after particle selection and was set for low resolution (CTF Autoprocessing).
Resolution: 17.6 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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