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- EMDB-10658: CryoEM structure of the ring-shaped virulence factor EspB from My... -

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Basic information

Entry
Database: EMDB / ID: EMD-10658
TitleCryoEM structure of the ring-shaped virulence factor EspB from Mycobacterium tuberculosis
Map dataSharped map
Sample
  • Complex: EspB Heptameric complex
    • Protein or peptide: ESX-1 secretion-associated protein EspB
KeywordsM. tuberculosis / ESX-1 / Type VII secretion system / EspB / Rv3881c / EsxA / CFP10 / ESAT-6 / TRANSPORT 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 H37Rv (bacteria) / Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.37 Å
AuthorsPiton J / Pojer F
Funding support Switzerland, 1 items
OrganizationGrant numberCountry
Swiss National Science Foundation31003A_162641 Switzerland
CitationJournal: J Struct Biol X / Year: 2020
Title: High resolution CryoEM structure of the ring-shaped virulence factor EspB from .
Authors: Jérémie Piton / Florence Pojer / Soichi Wakatsuki / Cornelius Gati / Stewart T Cole /
Abstract: The EspB protein of is a 60 kDa virulence factor, implicated in conjugation and exported by the ESX-1 system of which it may also be a component. Previous attempts to obtain high-resolution maps of ...The EspB protein of is a 60 kDa virulence factor, implicated in conjugation and exported by the ESX-1 system of which it may also be a component. Previous attempts to obtain high-resolution maps of EspB by cryo-electron microscopic examination of single particles have been thwarted by severe orientation bias of the particles. This was overcome by using detergent as a surfactant thereby allowing reconstruction of the EspB structure at 3.37 Å resolution. The final structure revealed the N-terminal domain of EspB to be organized as a cylindrical heptamer with dimensions of 90 Å x 90 Å and a central channel of 45 Å diameter whereas the C-terminal domain was unstructured. New atomic insight was obtained into the helical packing required for protomer interactions and the overall electrostatic potential. The external surface is electronegatively charged while the channel is lined with electropositive patches. EspB thus has many features of a pore-like transport protein that might allow the passage of an ESX-1 substrate such as the 35 Å diameter EsxA-EsxB heterodimer or B-form DNA consistent with its proposed role in DNA uptake.
History
DepositionFeb 3, 2020-
Header (metadata) releaseJun 17, 2020-
Map releaseJun 17, 2020-
UpdateMay 22, 2024-
Current statusMay 22, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6xzc
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10658.png

Downloads & links

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Map

FileDownload / File: emd_10658.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharped map
Voxel sizeX=Y=Z: 1.058 Å
Density
Contour LevelBy AUTHOR: 0.8 / Movie #1: 0.8
Minimum - Maximum-2.6964684 - 5.1885586
Average (Standard dev.)0.00056320796 (±0.10025215)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 423.19998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0581.0581.058
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z423.200423.200423.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-2.6965.1890.001

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Supplemental data

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Mask #1

Fileemd_10658_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Map A

Fileemd_10658_half_map_1.map
AnnotationMap A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Map B

Fileemd_10658_half_map_2.map
AnnotationMap B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : EspB Heptameric complex

EntireName: EspB Heptameric complex
Components
  • Complex: EspB Heptameric complex
    • Protein or peptide: ESX-1 secretion-associated protein EspB

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Supramolecule #1: EspB Heptameric complex

SupramoleculeName: EspB Heptameric complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Molecular weightTheoretical: 329 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 tuberculosis (strain ATCC 25618 / H37Rv) (bacteria)
Strain: ATCC 25618 / H37Rv
Molecular weightTheoretical: 47.637527 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MTQSQTVTVD QQEILNRANE VEAPMADPPT DVPITPCELT AAKNAAQQLV LSADNMREYL AAGAKERQRL ATSLRNAAKA YGEVDEEAA TALDNDGEGT VQAESAGAVG GDSSAELTDT PRVATAGEPN FMDLKEAARK LETGDQGASL AHFADGWNTF N LTLQGDVK ...String:
MTQSQTVTVD QQEILNRANE VEAPMADPPT DVPITPCELT AAKNAAQQLV LSADNMREYL AAGAKERQRL ATSLRNAAKA YGEVDEEAA TALDNDGEGT VQAESAGAVG GDSSAELTDT PRVATAGEPN FMDLKEAARK LETGDQGASL AHFADGWNTF N LTLQGDVK RFRGFDNWEG DAATACEASL DQQRQWILHM AKLSAAMAKQ AQYVAQLHVW ARREHPTYED IVGLERLYAE NP SARDQIL PVYAEYQQRS EKVLTEYNNK AALEPVNPPK PPPAIKIDPP PPPQEQGLIP GFLMPPSDGS GVTPGTGMPA APM VPPTGS PGGGLPADTA AQLTSAGREA AALSGDVAVK AASLGGGGGG GVPSAPLGSA IGGAESVRPA GAGDIAGLGQ GRAG GGAAL GGGGMGMPMG AAHQGQGGAK SKGSQQEDEA LYTEDRAWTE AVIGNRRRQD SKESK

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

Concentration5 mg/mL
BufferpH: 8 / Component:
ConcentrationFormula
20.0 mMTris-HCl
150.0 mMNaCl
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 10 sec.
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV
Details: 0.05% fluorinated octylmaltoside was add into protein solution and immediately applied to a freshly glow-discharged holey carbon grid. ). Excess liquid was blotted for 2.3 s using an FEI ...Details: 0.05% fluorinated octylmaltoside was add into protein solution and immediately applied to a freshly glow-discharged holey carbon grid. ). Excess liquid was blotted for 2.3 s using an FEI Vitrobot Mark IV and the sample was plunge frozen in liquid ethane..
DetailsThis sample was monodiperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number real images: 2600 / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 3.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 292964
Startup modelType of model: INSILICO MODEL
Final reconstructionNumber classes used: 50 / Applied symmetry - Point group: C7 (7 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.37 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.0) / Number images used: 143350
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: cryoSPARC

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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: FLEXIBLE FIT
Output model

PDB-6xzc:
CryoEM structure of the ring-shaped virulence factor EspB from Mycobacterium tuberculosis

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