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- EMDB-6120: Structure of a mycobacterial protein -

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Basic information

Entry
Database: EMDB / ID: EMD-6120
TitleStructure of a mycobacterial protein
Map dataReconstruction of mycobacterial protein
Sample
  • Sample: Mycobacterial protein
  • Protein or peptide: Mycobacterial protein
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 (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 30.0 Å
AuthorsSolomonson M / Setiaputra D / Makepeace KAT / Lameignere E / Petrotchenko EV / Conrady DG / Bergeron JR / Vuckovic M / DiMaio F / Borchers CH ...Solomonson M / Setiaputra D / Makepeace KAT / Lameignere E / Petrotchenko EV / Conrady DG / Bergeron JR / Vuckovic M / DiMaio F / Borchers CH / Yip CK / Strynadka NCJ
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, 2014-
Header (metadata) releaseNov 5, 2014-
Map releaseMar 11, 2015-
UpdateMar 18, 2015-
Current statusMar 18, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.7
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.7
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j83
  • Surface level: 0.7
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6120.gif

Downloads & links

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Map

FileDownload / File: emd_6120.map.gz / Format: CCP4 / Size: 1001 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of mycobacterial protein
Voxel sizeX=Y=Z: 4.7 Å
Density
Contour LevelBy AUTHOR: 0.7 / Movie #1: 0.7
Minimum - Maximum-2.66882896 - 1.93457854
Average (Standard dev.)-0.01802388 (±0.19855918)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions646464
Spacing646464
CellA=B=C: 300.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.74.74.7
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z300.800300.800300.800
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ969680
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS646464
D min/max/mean-2.6691.935-0.018

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Supplemental data

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Sample components

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Entire : Mycobacterial protein

EntireName: Mycobacterial protein
Components
  • Sample: Mycobacterial protein
  • Protein or peptide: Mycobacterial protein

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Supramolecule #1000: Mycobacterial protein

SupramoleculeName: Mycobacterial protein / type: sample / ID: 1000 / Number unique components: 1
Molecular weightExperimental: 230 KDa / Theoretical: 230 KDa
Method: size exclusion chromatography coupled to multi-angle light scattering

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Macromolecule #1: Mycobacterial protein

MacromoleculeName: Mycobacterial protein / type: protein_or_peptide / ID: 1 / Recombinant expression: Yes
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 / Recombinant plasmid: pET28a

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.01 mg/mL
BufferpH: 7.5 / Details: 20 mM HEPES, 150 mM NaCl
StainingType: NEGATIVE
Details: Protein was adsorbed onto grid, quickly blotted, and floated on 1% uranyl formate solution for 30 seconds
GridDetails: 200 mesh gold grid with thin carbon support, glow discharged
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 65900 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 49000
Sample stageSpecimen holder: side entry room temperature tomography holder
Specimen holder model: SIDE ENTRY, EUCENTRIC
TemperatureAverage: 298 K
DetailsLow dose
DateAug 28, 2013
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Digitization - Sampling interval: 1.3 µm / Number real images: 50 / Average electron dose: 25 e/Å2 / Bits/pixel: 16
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Final two d classificationNumber classes: 2
Final reconstructionResolution.type: BY AUTHOR / Resolution: 30.0 Å / Resolution method: OTHER / Software - Name: Spider, EMAN2
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.
Number images used: 1455

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Atomic model buiding 1

Initial modelPDB ID:

4wj1


Chain - Chain ID: A
SoftwareName: Rosetta
DetailsHomology model generated from PDB coordinates and used for symmetric docking in Rosetta.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: RMSD / Rosetta score
Output model

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

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