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- EMDB-1978: 3D structure of the Yersinia entomophaga toxin complex and implic... -

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Basic information

Entry
Database: EMDB / ID: EMD-1978
Title3D structure of the Yersinia entomophaga toxin complex and implications for insecticidal activity
Map dataSPA map of Y.entomophaga MH96(deletion construct 9) insecticidal Tc (Yen-Tc K9).
Sample
  • Sample: Insecticidal Tc from Yersinia entomophaga strain MH96 (deletion construct 9)(aka Yen-Tc K9)
  • Protein or peptide: Yen-Tc A1 subunit
  • Protein or peptide: Yen-Tc A2 subunit
  • Protein or peptide: Chi1
  • Protein or peptide: Chi2
Keywordstoxin complex / bacterial toxin / bioinsecticide
Function / homology
Function and homology information


chitinase / chitinase activity / chitin catabolic process / chitin binding / polysaccharide catabolic process / extracellular region
Similarity search - Function
Chitinase insertion domain superfamily / : / Chitinase II / Glyco_18 / Glycosyl hydrolases family 18 (GH18) domain profile. / Glycosyl hydrolases family 18 / Glycoside hydrolase family 18, catalytic domain / Glycoside hydrolase superfamily
Similarity search - Domain/homology
Biological speciesYersinia entomophaga (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 17.0 Å
AuthorsLandsberg MJ / Jones SA / Rothnagel R / Busby JN / Marshall SDG / Simpson RM / Lott JS / Hankamer B / Hurst MRH
CitationJournal: J Mol Biol / Year: 2012
Title: Structural analysis of Chi1 Chitinase from Yen-Tc: the multisubunit insecticidal ABC toxin complex of Yersinia entomophaga.
Authors: Jason N Busby / Michael J Landsberg / Robert M Simpson / Sandra A Jones / Ben Hankamer / Mark R H Hurst / J Shaun Lott /
Abstract: Yersinia entomophaga MH96 is a native New Zealand soil bacterium that secretes a large ABC-type protein toxin complex, Yen-Tc, similar to those produced by nematode-associated bacteria such as ...Yersinia entomophaga MH96 is a native New Zealand soil bacterium that secretes a large ABC-type protein toxin complex, Yen-Tc, similar to those produced by nematode-associated bacteria such as Photorhabdus luminescens. Y. entomophaga displays an exceptionally virulent pathogenic phenotype in sensitive insect species, causing death within 72 h of infection. Because of this phenotype, there is intrinsic interest in the mechanism of action of Yen-Tc, and it also has the potential to function as a novel class of biopesticide. We have identified genes that encode chitinases as part of the toxin complex loci in Y. entomophaga MH96, P. luminescens, Photorhabdus asymbiotica and Xenorhabdus nematophila. Furthermore, we have shown that the secreted toxin complex from Y. entomophaga MH96 includes two chitinases as an integral part of the complex, a feature not described previously in other ABC toxins and possibly related to the severe disease caused by this bacterium. We present here the structure of the Y. entomophaga MH96 Chi1 chitinase, determined by X-ray crystallography to 1.74 Å resolution, and show that a ring of five symmetrically arranged lobes on the surface of the Yen-Tc toxin complex structure, as determined by single-particle electron microscopy, provides a good fit to the Chi1 monomer. We also confirm that the isolated chitinases display endochitinase activity, as does the complete toxin complex.
History
DepositionOct 26, 2011-
Header (metadata) releaseNov 18, 2011-
Map releaseJan 20, 2012-
UpdateFeb 23, 2012-
Current statusFeb 23, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 6.13
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 6.13
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4a5q
  • Surface level: 6.13
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4a5q
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-1978.jpg

Downloads & links

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Map

FileDownload / File: emd_1978.map.gz / Format: CCP4 / Size: 21.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSPA map of Y.entomophaga MH96(deletion construct 9) insecticidal Tc (Yen-Tc K9).
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.9 Å/pix.
x 180 pix.
= 702. Å		3.9 Å/pix.
x 180 pix.
= 702. Å		3.9 Å/pix.
x 180 pix.
= 702. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.9 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 6.13 / Movie #1: 6.13
Minimum - Maximum-11.993600000000001 - 13.0684
Average (Standard dev.)0.00000000175681 (±0.871901)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-108-108-108
Dimensions180180180
Spacing180180180
CellA=B=C: 702 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.93.93.9
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z702.000702.000702.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-56-56-55
NX/NY/NZ112112112
MAP C/R/S123
start NC/NR/NS-108-108-108
NC/NR/NS180180180
D min/max/mean-11.99413.0680.000

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Supplemental data

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Sample components

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Entire : Insecticidal Tc from Yersinia entomophaga strain MH96 (deletion c...

EntireName: Insecticidal Tc from Yersinia entomophaga strain MH96 (deletion construct 9)(aka Yen-Tc K9)
Components
  • Sample: Insecticidal Tc from Yersinia entomophaga strain MH96 (deletion construct 9)(aka Yen-Tc K9)
  • Protein or peptide: Yen-Tc A1 subunit
  • Protein or peptide: Yen-Tc A2 subunit
  • Protein or peptide: Chi1
  • Protein or peptide: Chi2

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Supramolecule #1000: Insecticidal Tc from Yersinia entomophaga strain MH96 (deletion c...

SupramoleculeName: Insecticidal Tc from Yersinia entomophaga strain MH96 (deletion construct 9)(aka Yen-Tc K9)
type: sample / ID: 1000 / Details: Sample was monodisperse
Oligomeric state: 20mer comprising 5 A1, 5 A2, 5 Chi1 and 5 Chi2 subunits respectively.
Number unique components: 4
Molecular weightExperimental: 2.1 MDa / Theoretical: 2.1 MDa / Method: Dynamic light scattering

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Macromolecule #1: Yen-Tc A1 subunit

MacromoleculeName: Yen-Tc A1 subunit / type: protein_or_peptide / ID: 1 / Name.synonym: Yen-Tc A1 subunit / Number of copies: 5 / Oligomeric state: Pentamer / Recombinant expression: No
Source (natural)Organism: Yersinia entomophaga (bacteria) / Strain: MH96::9 / Organelle: Secreted
Molecular weightTheoretical: 130 KDa

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Macromolecule #2: Yen-Tc A2 subunit

MacromoleculeName: Yen-Tc A2 subunit / type: protein_or_peptide / ID: 2 / Name.synonym: Yen-Tc A2 subunit / Number of copies: 5 / Oligomeric state: Pentamer / Recombinant expression: No
Source (natural)Organism: Yersinia entomophaga (bacteria) / Strain: MH96::9 / Organelle: Secreted
Molecular weightTheoretical: 156 KDa

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Macromolecule #3: Chi1

MacromoleculeName: Chi1 / type: protein_or_peptide / ID: 3 / Name.synonym: Chi1 / Number of copies: 5 / Oligomeric state: Pentamer / Recombinant expression: No
Source (natural)Organism: Yersinia entomophaga (bacteria) / Strain: MH96::9 / Organelle: Secreted
Molecular weightTheoretical: 60 KDa

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Macromolecule #4: Chi2

MacromoleculeName: Chi2 / type: protein_or_peptide / ID: 4 / Name.synonym: Chi2 / Number of copies: 5 / Oligomeric state: Pentamer / Recombinant expression: No
Source (natural)Organism: Yersinia entomophaga (bacteria) / Strain: MH96::9 / Organelle: Secreted
Molecular weightTheoretical: 70 KDa

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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

Concentration0.1 mg/mL
BufferpH: 7.5 / Details: 25 mM Tris, 130 mM NaCl
StainingType: NEGATIVE
Details: Protein pre-absorbed onto carbon-coated grids and stained with 0.75% uranyl formate.
GridDetails: 400 mesh Cu grids
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F30
TemperatureAverage: 295 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 93,000 times magnification
DateJun 2, 2009
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 300 / Average electron dose: 80 e/Å2
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 0.95 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 59000
Sample stageSpecimen holder: FEI single tilt / Specimen holder model: SIDE ENTRY, EUCENTRIC
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

DetailsParticles were selected using semi-automated particle selection (SwarmPS,e2boxer.py)
Final reconstructionApplied symmetry - Point group: C5 (5 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Xmipp, EMAN / Number images used: 10604
Final two d classificationNumber classes: 403

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Atomic model buiding 1

Initial modelPDB ID:

3oa5


Chain - Chain ID: A
SoftwareName: SITUS
DetailsPDBEntryID_givenInChain. Protocol: Rigid Body. Colores was used to perform an exhaustive 6d search. A Laplacian filter was used to emphasise correct contour fitting.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 22.09 / Target criteria: Cross-correlation coefficient
Output model

PDB-4a5q:
Crystal structure of the chitinase Chi1 fitted into the 3D structure of the Yersinia entomophaga toxin complex

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