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- EMDB-2417: Negative staining 3D-EM of the progenitor toxin complex (PTC) of ... -

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Entry
Database: EMDB / ID: EMD-2417
TitleNegative staining 3D-EM of the progenitor toxin complex (PTC) of Botulinum toxin type A
Map datanegative staining 3D-EM of the the progenitor toxin complex (PTC) of Botulinum toxin type A
Sample
  • Sample: The progenitor toxin complexes of Botulinum toxin type A
  • Protein or peptide: BoNT/A
  • Protein or peptide: NTNHA
  • Protein or peptide: HA17
  • Protein or peptide: HA33
  • Protein or peptide: HA70
KeywordsBotulinum toxin / the progenitor toxin complex (PTC)
Biological speciesClostridium botulinum (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 30.0 Å
AuthorsLee K / Gu S / Jin L / Le TTN / Cheng LW / Strotmeier J / Kruel AM / Yao G / Perry K / Rummel A / Jin R
CitationJournal: PLoS Pathog / Year: 2013
Title: Structure of a bimodular botulinum neurotoxin complex provides insights into its oral toxicity.
Authors: Kwangkook Lee / Shenyan Gu / Lei Jin / Thi Tuc Nghi Le / Luisa W Cheng / Jasmin Strotmeier / Anna Magdalena Kruel / Guorui Yao / Kay Perry / Andreas Rummel / Rongsheng Jin /
Abstract: Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and cause the fatal disease botulism, a flaccid paralysis of the muscle. BoNTs are released together with several auxiliary ...Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and cause the fatal disease botulism, a flaccid paralysis of the muscle. BoNTs are released together with several auxiliary proteins as progenitor toxin complexes (PTCs) to become highly potent oral poisons. Here, we report the structure of a ∼760 kDa 14-subunit large PTC of serotype A (L-PTC/A) and reveal insight into its absorption mechanism. Using a combination of X-ray crystallography, electron microscopy, and functional studies, we found that L-PTC/A consists of two structurally and functionally independent sub-complexes. A hetero-dimeric 290 kDa complex protects BoNT, while a hetero-dodecameric 470 kDa complex facilitates its absorption in the harsh environment of the gastrointestinal tract. BoNT absorption is mediated by nine glycan-binding sites on the dodecameric sub-complex that forms multivalent interactions with carbohydrate receptors on intestinal epithelial cells. We identified monosaccharides that blocked oral BoNT intoxication in mice, which suggests a new strategy for the development of preventive countermeasures for BoNTs based on carbohydrate receptor mimicry.
History
DepositionJul 17, 2013-
Header (metadata) releaseAug 7, 2013-
Map releaseOct 23, 2013-
UpdateOct 23, 2013-
Current statusOct 23, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 2.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2417_500...

Downloads & links

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Map

FileDownload / File: emd_2417.map.gz / Format: CCP4 / Size: 5.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationnegative staining 3D-EM of the the progenitor toxin complex (PTC) of Botulinum toxin type A
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.28 Å/pix.
x 112 pix.
= 479.36 Å		4.28 Å/pix.
x 112 pix.
= 479.36 Å		4.28 Å/pix.
x 112 pix.
= 479.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.28 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.5 / Movie #1: 2.5
Minimum - Maximum-0.15406871 - 32.892124180000003
Average (Standard dev.)0.0 (±1.33123147)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-56-56-56
Dimensions112112112
Spacing112112112
CellA=B=C: 479.36002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.284.284.28
M x/y/z112112112
origin x/y/z0.0000.0000.000
length x/y/z479.360479.360479.360
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS-56-56-56
NC/NR/NS112112112
D min/max/mean-0.15432.892-0.000

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Supplemental data

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Sample components

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Entire : The progenitor toxin complexes of Botulinum toxin type A

EntireName: The progenitor toxin complexes of Botulinum toxin type A
Components
  • Sample: The progenitor toxin complexes of Botulinum toxin type A
  • Protein or peptide: BoNT/A
  • Protein or peptide: NTNHA
  • Protein or peptide: HA17
  • Protein or peptide: HA33
  • Protein or peptide: HA70

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Supramolecule #1000: The progenitor toxin complexes of Botulinum toxin type A

SupramoleculeName: The progenitor toxin complexes of Botulinum toxin type A
type: sample / ID: 1000
Details: The complex is monodisperse, and stable only at acidic pH.
Oligomeric state: A 14-subunit complex of 1 BoNT/A, 1 NTNHA, 3 HA70, 3 HA17 and 6 HA33
Number unique components: 5
Molecular weightExperimental: 760 KDa / Theoretical: 760 KDa

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Macromolecule #1: BoNT/A

MacromoleculeName: BoNT/A / type: protein_or_peptide / ID: 1 / Recombinant expression: Yes
Source (natural)Organism: Clostridium botulinum (bacteria) / Location in cell: secreted
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21-RIL (DE3) / Recombinant plasmid: pQE30, pET28a, pRSFDuet-1

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Macromolecule #2: NTNHA

MacromoleculeName: NTNHA / type: protein_or_peptide / ID: 2 / Recombinant expression: Yes
Source (natural)Organism: Clostridium botulinum (bacteria) / Location in cell: secreted
Molecular weightTheoretical: 760 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21-RIL (DE3) / Recombinant plasmid: pQE30, pET28a, pRSFDuet-1

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Macromolecule #3: HA17

MacromoleculeName: HA17 / type: protein_or_peptide / ID: 3 / Recombinant expression: Yes
Source (natural)Organism: Clostridium botulinum (bacteria) / Location in cell: secreted
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21-RIL (DE3) / Recombinant plasmid: pQE30, pET28a, pRSFDuet-1

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Macromolecule #4: HA33

MacromoleculeName: HA33 / type: protein_or_peptide / ID: 4 / Recombinant expression: Yes
Source (natural)Organism: Clostridium botulinum (bacteria) / Location in cell: secreted
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21-RIL (DE3) / Recombinant plasmid: pQE30, pET28a, pRSFDuet-1

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Macromolecule #5: HA70

MacromoleculeName: HA70 / type: protein_or_peptide / ID: 5 / Recombinant expression: Yes
Source (natural)Organism: Clostridium botulinum (bacteria) / Location in cell: secreted
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21-RIL (DE3) / Recombinant plasmid: pQE30, pET28a, pRSFDuet-1

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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

Concentration0.02 mg/mL
BufferpH: 6.2 / Details: 100mM NaCl, 20mM MES
StainingType: NEGATIVE
Details: Grids with adsorbed protein floated on 1% w/v uranyl formate for 30 seconds
GridDetails: 300 mesh copper grid with carbon support, glow discharged in vacuumed air
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 70,000 times magnification.
DateFeb 4, 2012
Image recordingCategory: CCD / Film or detector model: GENERIC TVIPS (4k x 4k) / Number real images: 362 / Average electron dose: 40 e/Å2 / Bits/pixel: 8
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 70000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 3.6 µm / Nominal defocus min: 1.5 µm
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsImage processing was done using EMAN2.The particles were selected by a semi-automatic selection using e2boxer.
CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 30.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN2 / Number images used: 15140

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