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- PDB-3j83: Heptameric EspB Rosetta model guided by EM density -

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Basic information

Entry
Database: PDB / ID: 3j83
TitleHeptameric EspB Rosetta model guided by EM density
ComponentsESX-1 secretion-associated protein EspB
KeywordsPROTEIN BINDING
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 / negative staining / Resolution: 30 Å
AuthorsSolomonson, M. / DiMaio, F. / Strynadka, N.C.J.
CitationJournal: Structure / Year: 2015
Title: Structure of EspB from the ESX-1 type VII secretion system and insights into its export mechanism.
Authors: Matthew Solomonson / Dheva Setiaputra / Karl A T Makepeace / Emilie Lameignere / Evgeniy V Petrotchenko / Deborah G Conrady / Julien R Bergeron / Marija Vuckovic / Frank DiMaio / Christoph H ...Authors: Matthew Solomonson / Dheva Setiaputra / Karl A T Makepeace / Emilie Lameignere / Evgeniy V Petrotchenko / Deborah G Conrady / Julien R Bergeron / Marija Vuckovic / Frank DiMaio / Christoph H Borchers / Calvin K Yip / Natalie C J Strynadka /
Abstract: Mycobacterium tuberculosis (Mtb) uses the ESX-1 type VII secretion system to export virulence proteins across its lipid-rich cell wall, which helps permeabilize the host's macrophage phagosomal ...Mycobacterium tuberculosis (Mtb) uses the ESX-1 type VII secretion system to export virulence proteins across its lipid-rich cell wall, which helps permeabilize the host's macrophage phagosomal membrane, facilitating the escape and cell-to-cell spread of Mtb. ESX-1 membranolytic activity depends on a set of specialized secreted Esp proteins, the structure and specific roles of which are not currently understood. Here, we report the X-ray and electron microscopic structures of the ESX-1-secreted EspB. We demonstrate that EspB adopts a PE/PPE-like fold that mediates oligomerization with apparent heptameric symmetry, generating a barrel-shaped structure with a central pore that we propose contributes to the macrophage killing functions of EspB. Our structural data also reveal unexpected direct interactions between the EspB bipartite secretion signal sequence elements that form a unified aromatic surface. These findings provide insight into how specialized proteins encoded within the ESX-1 locus are targeted for secretion, and for the first time indicate an oligomerization-dependent role for Esp virulence factors.
History
DepositionSep 30, 2014Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 11, 2015Provider: repository / Type: Initial release
Revision 1.1Mar 18, 2015Group: Database references
Revision 1.2Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.image_processing_id
Revision 1.3Feb 21, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _struct_ref_seq_dif.details

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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)276,8837
Polymers276,8837
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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


Mass: 39554.656 Da / Num. of mol.: 7 / Fragment: UNP residues 1-348
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Gene: espB, mtb48, Rv3881c, MTV027.16c / 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: Mycobacterial protein / Type: COMPLEX
Molecular weightValue: 0.23 MDa / Experimental value: YES
Buffer solutionName: 20 mM HEPES, pH 7.5, 150 mM NaCl / pH: 7.5 / Details: 20 mM HEPES, pH 7.5, 150 mM NaCl
SpecimenConc.: 0.01 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: NO
Details: Protein was adsorbed onto grid, quickly blotted, and floated on 1% uranyl formate solution for 30 seconds.
EM stainingType: NEGATIVE / Material: uranyl formate
Specimen supportDetails: 200 mesh gold grid with thin carbon support, glow discharged

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI SPIRIT / Date: Aug 28, 2013 / Details: Low dose
Electron gunElectron source: LAB6 / Accelerating voltage: 120 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 49000 X / Calibrated magnification: 65900 X / Nominal defocus max: 1200 nm / Nominal defocus min: 800 nm / Camera length: 0 mm
Specimen holderSpecimen holder model: SIDE ENTRY, EUCENTRIC
Specimen holder type: side entry room temperature tomography holder
Temperature: 298 K
Image recordingElectron dose: 25 e/Å2 / Film or detector model: FEI EAGLE (4k x 4k)
Image scansNum. digital images: 50

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Processing

EM software
IDNameVersionCategory
1Rosettamodel fitting
2EMAN23D reconstruction
3SPIDER3D reconstruction
SymmetryPoint symmetry: C7 (7 fold cyclic)
3D reconstructionResolution: 30 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 1455 / Nominal pixel size: 4.7 Å / Actual pixel size: 4.7 Å
Details: Initial reconstructions were determined from a side-view 2D average based on observed 7-fold symmetry of the top view using a rotational extrusion method. The initial model was then refined ...Details: Initial reconstructions were determined from a side-view 2D average based on observed 7-fold symmetry of the top view using a rotational extrusion method. The initial model was then refined in 6 iterations with EMAN2 using untilted particles. (Single particle--Applied symmetry: C7)
Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: RMSD / Rosetta score
Details: REFINEMENT PROTOCOL--rigid body DETAILS--Homology model generated from PDB coordinates and used for symmetric docking in Rosetta.
Atomic model buildingPDB-ID: 4WJ1

4wj1


Pdb chain-ID: A / Accession code: 4WJ1 / Source name: PDB / Type: experimental model
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms14826 0 0 0 14826

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