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- EMDB-13153: 2.43 A Mycobacterium marinum EspB. -

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Basic information

Entry
Database: EMDB / ID: EMD-13153
Title2.43 A Mycobacterium marinum EspB.
Map dataLocal B-factor sharpened map from LocSpiral
Sample
  • Complex: Heptamer of EspB
    • Protein or peptide: ESX-1 secretion-associated protein EspB
KeywordsCryo-EM / EspB / ESX-1 / Preferential orientation / PROTEIN TRANSPORT
Function / homologyESX-1 secretion-associated protein EspB, PE domain / : / ESX-1 secreted protein B PE domain / ESX-1 secretion-associated protein EspB, PPE domain / PPE superfamily / extracellular region / ESX-1 secretion-associated protein EspB
Function and homology information
Biological speciesMycobacterium marinum (bacteria) / Mycobacterium marinum (strain ATCC BAA-535 / M) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.43 Å
AuthorsGijsbers A / Zhang Y
Funding support Netherlands, European Union, Mexico, 4 items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)731.016.407 Netherlands
Netherlands Organisation for Scientific Research (NWO)184.034.014 Netherlands
European Union (EU)No 766970 Q-SORTEuropean Union
Consejo Nacional de Ciencia y Tecnologia (CONACYT)283909 Mexico
CitationJournal: Curr Res Struct Biol / Year: 2021
Title: Priming mycobacterial ESX-secreted protein B to form a channel-like structure.
Authors: Abril Gijsbers / Vanesa Vinciauskaite / Axel Siroy / Ye Gao / Giancarlo Tria / Anjusha Mathew / Nuria Sánchez-Puig / Carmen López-Iglesias / Peter J Peters / Raimond B G Ravelli /
Abstract: ESX-1 is a major virulence factor of , a secretion machinery directly involved in the survival of the microorganism from the immune system defence. It disrupts the phagosome membrane of the host cell ...ESX-1 is a major virulence factor of , a secretion machinery directly involved in the survival of the microorganism from the immune system defence. It disrupts the phagosome membrane of the host cell through a contact-dependent mechanism. Recently, the structure of the inner-membrane core complex of the homologous ESX-3 and ESX-5 was resolved; however, the elements involved in the secretion through the outer membrane or those acting on the host cell membrane are unknown. Protein substrates might form this missing element. Here, we describe the oligomerisation process of the ESX-1 substrate EspB, which occurs upon cleavage of its C-terminal region and is favoured by an acidic environment. Cryo-electron microscopy data shows that quaternary structure of EspB is conserved across slow growing species, but not in the fast growing . EspB assembles into a channel with dimensions and characteristics suitable for the transit of ESX-1 substrates, as shown by the presence of another EspB trapped within. Our results provide insight into the structure and assembly of EspB, and suggests a possible function as a structural element of ESX-1.
History
DepositionJul 1, 2021-
Header (metadata) releaseAug 18, 2021-
Map releaseAug 18, 2021-
UpdateJul 17, 2024-
Current statusJul 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 6
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 6
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7p0z
  • Surface level: 6
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13153.png

Downloads & links

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Map

FileDownload / File: emd_13153.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocal B-factor sharpened map from LocSpiral
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 256 pix.
= 213.504 Å		0.83 Å/pix.
x 256 pix.
= 213.504 Å		0.83 Å/pix.
x 256 pix.
= 213.504 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.834 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 6.0 / Movie #1: 6
Minimum - Maximum0.0 - 23.074642000000001
Average (Standard dev.)0.2460945 (±1.0260041)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 213.504 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8340.8340.834
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z213.504213.504213.504
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ500500500
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean0.00023.0750.246

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Supplemental data

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Mask #1

Fileemd_13153_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Global b-factor sharpened map from Relion postprocess.

Fileemd_13153_additional_1.map
AnnotationGlobal b-factor sharpened map from Relion postprocess.
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unmasked halfmap 1 from Refine3D

Fileemd_13153_half_map_1.map
AnnotationUnmasked halfmap 1 from Refine3D
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unmasked halfmap 2 from Refine3D

Fileemd_13153_half_map_2.map
AnnotationUnmasked halfmap 2 from Refine3D
Projections & Slices
AxesZYX

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Histogram

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Sample components

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Entire : Heptamer of EspB

EntireName: Heptamer of EspB
Components
  • Complex: Heptamer of EspB
    • Protein or peptide: ESX-1 secretion-associated protein EspB

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Supramolecule #1: Heptamer of EspB

SupramoleculeName: Heptamer of EspB / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mycobacterium marinum (bacteria) / Strain: BAA-535
Molecular weightTheoretical: 210 KDa

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Macromolecule #1: ESX-1 secretion-associated protein EspB

MacromoleculeName: ESX-1 secretion-associated protein EspB / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO
Source (natural)Organism: Mycobacterium marinum (strain ATCC BAA-535 / M) (bacteria)
Strain: ATCC BAA-535 / M
Molecular weightTheoretical: 31.274275 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SMHSQPQTVT VDQQEILNRA DEVEAPMATP PTDVPQAPSG LTAANNAAEQ LAVSADNVRL YLQAGERERQ RLATSLRNAA AAYGEVEDE SATALDNDGN GEVDAQSAGG AGAGQTESLE ETPKVAAAGE SDFTDLKTAA TKLESGDQGT SMVNFADGWN N FNLSLQRD ...String:
SMHSQPQTVT VDQQEILNRA DEVEAPMATP PTDVPQAPSG LTAANNAAEQ LAVSADNVRL YLQAGERERQ RLATSLRNAA AAYGEVEDE SATALDNDGN GEVDAQSAGG AGAGQTESLE ETPKVAAAGE SDFTDLKTAA TKLESGDQGT SMVNFADGWN N FNLSLQRD IKRFRIFENW EGDAATACEA SMDQQKEWIL HMAKLSASLA KQANFMAQLQ LWARRGHPTL ADIVELERLA KD PDYQEQA IKLYAEYQET SEKVLSEYNT KADLEPVNPP KPPAAIKIDP P

UniProtKB: ESX-1 secretion-associated protein EspB

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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

Concentration8.7 mg/mL
BufferpH: 5.5
Component:
ConcentrationFormulaName
20.0 mMCH3COOHAcetate
150.0 mMNaClSodium Chloride
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 50 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 40 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeTFS KRIOS
TemperatureMin: 90.0 K
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
DetailsBasic direct alignments were done as well as astigmatism and coma alignment using AutoCTF
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 2421 / Average exposure time: 1.8 sec. / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 105000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: -2.0 µm / Nominal defocus min: -1.25 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4xxx

Final reconstructionApplied symmetry - Point group: C7 (7 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.43 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 435505
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4xxx


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 45.53 / Target criteria: Correlation coefficient
Output model

PDB-7p0z:
2.43 A Mycobacterium marinum EspB.

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