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- PDB-7p13: 2.29 A Mycobacterium tuberculosis EspB. -

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Basic information

Entry
Database: PDB / ID: 7p13
Title2.29 A Mycobacterium tuberculosis 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 / protein secretion by the type VII secretion system / PPE superfamily / biological process involved in interaction with host / extracellular region / identical protein binding / ESX-1 secretion-associated protein EspB
Function and homology information
Biological speciesMycobacterium tuberculosis H37RV (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.29 Å
AuthorsGijsbers, A. / Zhang, Y. / Vinciauskaite, V. / Siroy, A. / Gao, Y. / 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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Structure viewerMolecule:
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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)220,9337
Polymers220,9337
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry, Native mass spectrometry, data present in the manuscript, SAXS, Data not present in this manuscript, gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area11580 Å2
ΔGint40 kcal/mol
Surface area85130 Å2
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "F"
d_2ens_1chain "B"
d_3ens_1chain "C"
d_4ens_1chain "D"
d_5ens_1chain "E"
d_6ens_1chain "A"
d_7ens_1chain "G"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1VALPROF1 - 249
d_21ens_1VALPROB1 - 249
d_31ens_1VALPROC1 - 249
d_41ens_1VALPROD1 - 249
d_51ens_1VALPROE1 - 249
d_61ens_1VALPROA1 - 249
d_71ens_1VALPROG1 - 249

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Components

#1: Protein
ESX-1 secretion-associated protein EspB / Antigen MTB48


Mass: 31561.842 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37RV (bacteria)
Plasmid: pAG04 / Production host: Escherichia coli (E. coli) / References: UniProt: P9WJD9

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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: 0.22 MDa / Experimental value: YES
Source (natural)Organism: Mycobacterium tuberculosis H37RV (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Plasmid: pGA04
Buffer solutionpH: 5.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMAcetateCH3COOH1
2150 mMSodium ChlorideNaClSodium chloride1
SpecimenConc.: 6 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 / Cs: 2.7 mm / C2 aperture diameter: 50 µm
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: 2334
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
2EPU2.6.1image acquisition
4Gctf1.06CTF correction
7Coot0.9.4model fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
13PHENIX1.18.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 484786
SymmetryPoint symmetry: C7 (7 fold cyclic)
3D reconstructionResolution: 2.29 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 484786 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingB value: 32.59 / 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: 41.58 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.015913853
ELECTRON MICROSCOPYf_angle_d1.053818851
ELECTRON MICROSCOPYf_chiral_restr0.06722051
ELECTRON MICROSCOPYf_plane_restr0.00752555
ELECTRON MICROSCOPYf_dihedral_angle_d5.51771883
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2FELECTRON MICROSCOPYNCS constraints0.000625080737231
ens_1d_3FELECTRON MICROSCOPYNCS constraints0.000584231523429
ens_1d_4FELECTRON MICROSCOPYNCS constraints0.000712222759193
ens_1d_5FELECTRON MICROSCOPYNCS constraints0.00071443179787
ens_1d_6FELECTRON MICROSCOPYNCS constraints0.000685268674874
ens_1d_7FELECTRON MICROSCOPYNCS constraints0.000631338483846

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