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- EMDB-33752: Cryo-EM structure of Mycobacterial Type VII Secretion System Viru... -

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Entry
Database: EMDB / ID: EMD-33752
TitleCryo-EM structure of Mycobacterial Type VII Secretion System Virulence Factor EspB (residues 1-332) with Phosphatidic acid (PA)
Map dataMycobacterial Type VII Secretion System virulence factor EspB with phosphatidic acid (PA)
Sample
  • Complex: Cryo-EM structure of Mycobacterial Type VII Secretion System Virulence Factor EspB (residues 1-332) with Phosphatidic acid (PA)
KeywordsMycobacterium tuberculosis / T7SS / cryo-EM / EspB / membrane / lipid / LIPID BINDING PROTEIN
Biological speciesMycobacterium tuberculosis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.6 Å
AuthorsSengupta N / Padmanaban S / Dutta S
Funding support India, 3 items
OrganizationGrant numberCountry
Department of Biotechnology (DBT, India)BT/INF/22/SP22844/2017 India
Department of Science & Technology (DST, India)SR/FST/LSII-039/2015 India
Department of Biotechnology (DBT, India)BT/PR25580/BRB/10/1619/2017 India
CitationJournal: J Biol Chem / Year: 2023
Title: Cryo-EM reveals the membrane-binding phenomenon of EspB, a virulence factor of the mycobacterial type VII secretion system.
Authors: Nayanika Sengupta / Surekha Padmanaban / Somnath Dutta /
Abstract: Mycobacterium tuberculosis (Mtb) utilizes sophisticated machinery called the type VII secretion system to translocate virulence factors across its complex lipid membrane. EspB, a ∼36 kDa secreted ...Mycobacterium tuberculosis (Mtb) utilizes sophisticated machinery called the type VII secretion system to translocate virulence factors across its complex lipid membrane. EspB, a ∼36 kDa secreted substrate of the ESX-1 apparatus, was shown to cause ESAT-6-independent host cell death. Despite the current wealth of high-resolution structural information of the ordered N-terminal domain, the mechanism of EspB-mediated virulence remains poorly characterized. Here, we document EspB interaction with phosphatidic acid (PA) and phosphatidylserine (PS) in the context of membranes, through a biophysical approach including transmission electron microscopy and cryo-EM. We were also able to show PA, PS-dependent conversion of monomers to oligomers at physiological pH. Our data suggest that EspB adheres to biological membranes with limited PA and PS. EM of yeast mitochondria with EspB indicates a mitochondrial membrane-binding property of this ESX-1 substrate. Further, we determined the 3D structures of EspB with and without PA and observed plausible stabilization of the low complexity C-terminal domain in the presence of PA. Collectively, our cryo-EM-based structural and functional studies of EspB provide further insight into the host-Mtb interaction.
History
DepositionJul 4, 2022-
Header (metadata) releaseMar 22, 2023-
Map releaseMar 22, 2023-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33752.png

Downloads & links

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Map

FileDownload / File: emd_33752.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMycobacterial Type VII Secretion System virulence factor EspB with phosphatidic acid (PA)
Voxel sizeX=Y=Z: 0.92 Å
Density
Contour LevelBy AUTHOR: 0.0117
Minimum - Maximum-0.049682084 - 0.07316708
Average (Standard dev.)0.00008847259 (±0.0031475553)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 220.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Mycobacterial Type VII Secretion System virulence factor EspB...

Fileemd_33752_half_map_1.map
AnnotationMycobacterial Type VII Secretion System virulence factor EspB with phosphatidic acid (PA) - half map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Mycobacterial Type VII Secretion System virulence factor EspB...

Fileemd_33752_half_map_2.map
AnnotationMycobacterial Type VII Secretion System virulence factor EspB with phosphatidic acid (PA) - half map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cryo-EM structure of Mycobacterial Type VII Secretion System Viru...

EntireName: Cryo-EM structure of Mycobacterial Type VII Secretion System Virulence Factor EspB (residues 1-332) with Phosphatidic acid (PA)
Components
  • Complex: Cryo-EM structure of Mycobacterial Type VII Secretion System Virulence Factor EspB (residues 1-332) with Phosphatidic acid (PA)

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Supramolecule #1: Cryo-EM structure of Mycobacterial Type VII Secretion System Viru...

SupramoleculeName: Cryo-EM structure of Mycobacterial Type VII Secretion System Virulence Factor EspB (residues 1-332) with Phosphatidic acid (PA)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus min: 0.75 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 2.0 µm
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C7 (7 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 6.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 117898
FSC plot (resolution estimation)

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