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- PDB-7p0z: 2.43 A Mycobacterium marinum EspB. -

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Basic information

Entry
Database: PDB / ID: 7p0z
Title2.43 A Mycobacterium marinum EspB.
ComponentsESX-1 secretion-associated protein EspB
KeywordsPROTEIN TRANSPORT / Cryo-EM / EspB / ESX-1 / Preferential orientation
Function / homologyESX-1 secretion-associated protein EspB, PE domain / ESX-1 secreted protein B PE domain / extracellular region / ESX-1 secretion-associated protein EspB
Function and homology information
Biological speciesMycobacterium marinum (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.43 Å
AuthorsGijsbers, A. / Zhang, Y. / Vinciauskaite, V. / Siroy, A. / Ye, G. / Tria, G. / Mathew, A. / Sanchez-Puig, N. / Lopez-Iglesias, C. / Peters, P.J. / Ravelli, R.B.G.
Funding support Netherlands, European Union, Mexico, 4items
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, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 18, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 13, 2021Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_database_proc
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
A: ESX-1 secretion-associated protein EspB
B: ESX-1 secretion-associated protein EspB
C: ESX-1 secretion-associated protein EspB
D: ESX-1 secretion-associated protein EspB
E: ESX-1 secretion-associated protein EspB
F: ESX-1 secretion-associated protein EspB
G: ESX-1 secretion-associated protein EspB


Theoretical massNumber of molelcules
Total (without water)218,9207
Polymers218,9207
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: assay for oligomerization, gel filtration, mass spectrometry, native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area12470 Å2
ΔGint-7 kcal/mol
Surface area79100 Å2
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "D"
d_2ens_1chain "B"
d_3ens_1chain "C"
d_4ens_1chain "G"
d_5ens_1chain "E"
d_6ens_1chain "F"
d_7ens_1chain "A"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1THRPROD1 - 236
d_21ens_1THRPROB1 - 236
d_31ens_1THRPROC1 - 236
d_41ens_1THRPROG1 - 236
d_51ens_1THRPROE1 - 236
d_61ens_1THRPROF1 - 236
d_71ens_1THRPROA1 - 236

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Components

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


Mass: 31274.275 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium marinum (strain ATCC BAA-535 / M) (bacteria)
Strain: ATCC BAA-535 / M / Gene: espB, MMAR_5457 / Plasmid: pAG10 / Production host: Escherichia coli (E. coli) / References: UniProt: B2HNQ9

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Heptamer of EspB / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.21 MDa / Experimental value: YES
Source (natural)Organism: Mycobacterium marinum (bacteria) / Strain: BAA-535
Source (recombinant)Organism: Escherichia coli (E. coli) / Plasmid: pAG10
Buffer solutionpH: 5.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMAcetateCH3COOH1
2150 mMSodium ChlorideNaClSodium chloride1
SpecimenConc.: 8.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Details: Basic direct alignments were done as well as astigmatism and coma alignment using AutoCTF
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Calibrated magnification: 105000 X / Nominal defocus max: -2000 nm / Nominal defocus min: -1250 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (min): 90 K
Image recordingAverage exposure time: 1.8 sec. / Electron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2421
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 5760 / Height: 4092

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18.2_3874refinement
PHENIX1.18.2_3874refinement
EM software
IDNameVersionCategory
1RELION3.1particle selection
2EPU2.6.1image acquisition
4Gctf1.06CTF correction
7Coot0.9.4model fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
19PHENIX1.18.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C7 (7 fold cyclic)
3D reconstructionResolution: 2.43 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 435505 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingB value: 45.53 / Protocol: OTHER / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 4XXX

4xxx


Pdb chain-ID: A
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 45.53 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.015613111
ELECTRON MICROSCOPYf_angle_d0.919517815
ELECTRON MICROSCOPYf_chiral_restr0.05881967
ELECTRON MICROSCOPYf_plane_restr0.00552401
ELECTRON MICROSCOPYf_dihedral_angle_d4.51721764
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2DELECTRON MICROSCOPYNCS constraints0.000707633448422
ens_1d_3DELECTRON MICROSCOPYNCS constraints0.00071966937074
ens_1d_4DELECTRON MICROSCOPYNCS constraints0.000709131042041
ens_1d_5DELECTRON MICROSCOPYNCS constraints0.000587441571166
ens_1d_6DELECTRON MICROSCOPYNCS constraints0.00072731787848
ens_1d_7DELECTRON MICROSCOPYNCS constraints0.000684206280918

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