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- EMDB-2416: Negative staining 3D-EM of the HA complex of Botulinum toxin type A -

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Basic information

Entry
Database: EMDB / ID: EMD-2416
TitleNegative staining 3D-EM of the HA complex of Botulinum toxin type A
Map datanegative staining 3D-EM of the HA complex of Botulinum toxin A
Sample
  • Sample: The trimeric HA complex of Botulinum toxin type A
  • Protein or peptide: Botulinum toxin type A HA17
  • Protein or peptide: Botulinum toxin type A HA33
  • Protein or peptide: Botulinum toxin type A HA70
KeywordsBotulinum toxin / HA complex
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: 0.55
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.55
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2416_500...

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Map

FileDownload / File: emd_2416.map.gz / Format: CCP4 / Size: 6.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationnegative staining 3D-EM of the HA complex of Botulinum toxin A
Voxel sizeX=Y=Z: 4.28 Å
Density
Contour LevelBy AUTHOR: 0.55 / Movie #1: 0.55
Minimum - Maximum-1.45518267 - 2.65945172
Average (Standard dev.)0.00273009 (±0.11532168)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-60-60-60
Dimensions120120120
Spacing120120120
CellA=B=C: 513.60004 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.284.284.28
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z513.600513.600513.600
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS-60-60-60
NC/NR/NS120120120
D min/max/mean-1.4552.6590.003

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Supplemental data

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Sample components

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Entire : The trimeric HA complex of Botulinum toxin type A

EntireName: The trimeric HA complex of Botulinum toxin type A
Components
  • Sample: The trimeric HA complex of Botulinum toxin type A
  • Protein or peptide: Botulinum toxin type A HA17
  • Protein or peptide: Botulinum toxin type A HA33
  • Protein or peptide: Botulinum toxin type A HA70

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Supramolecule #1000: The trimeric HA complex of Botulinum toxin type A

SupramoleculeName: The trimeric HA complex of Botulinum toxin type A / type: sample / ID: 1000
Details: The trimeric HA complex is monodisperse, and stable at both pH2.5 and pH7.4.
Oligomeric state: A trimer of 3 HA70, 3 HA17 and 6 HA33 / Number unique components: 3
Molecular weightExperimental: 470 KDa / Theoretical: 470 KDa / Method: Sedimentation

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Macromolecule #1: Botulinum toxin type A HA17

MacromoleculeName: Botulinum toxin type A HA17 / 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: Botulinum toxin type A HA33

MacromoleculeName: Botulinum toxin type A HA33 / type: protein_or_peptide / ID: 2 / 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 #3: Botulinum toxin type A HA70

MacromoleculeName: Botulinum toxin type A HA70 / 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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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: 7.6 / Details: 50mM NaCl, 20mM Tris-HCL
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
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 70000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 3.6 µm / Nominal defocus min: 1.5 µm
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 70,000 times magnification.
DateOct 4, 2011
Image recordingCategory: CCD / Film or detector model: GENERIC TVIPS (4k x 4k) / Number real images: 62 / Average electron dose: 40 e/Å2 / Bits/pixel: 8
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 30.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN2 / Details: C3 symmetry was imposed in refinement / Number images used: 3746
DetailsImage processing was done using EMAN2.The particles were selected by a semi-automatic selection using e2boxer.

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