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- PDB-6tfk: Vip3Aa toxin structure -

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Basic information

Entry
Database: PDB / ID: 6tfk
TitleVip3Aa toxin structure
ComponentsVegetative insecticidal protein
KeywordsTOXIN / Vip3Aa / beta prism / insecticidal protein / Vip3
Function / homologyVegetative insecticide protein 3 / Vegetative insecticide protein 3A N terminal / cellular anatomical entity / Carbohydrate-binding, CenC-like / Carbohydrate binding domain / hydrolase activity, acting on glycosyl bonds / Galactose-binding-like domain superfamily / Vegetative insecticidal protein
Function and homology information
Biological speciesBacillus thuringiensis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsNunez-Ramirez, R. / Huesa, J. / Bel, Y. / Ferre, J. / Casino, P. / Arias-Palomo, E.
Funding support Spain, 5items
OrganizationGrant numberCountry
Spanish Ministry of Economy and CompetitivenessBFU2017-89143-P Spain
Spanish Ministry of Economy and CompetitivenessRYC-2015-19059 Spain
Spanish Ministry of Economy and CompetitivenessBFU2016-78606-P Spain
Spanish Ministry of Economy and CompetitivenessRYC-2014-16490 Spain
Spanish Ministry of Science, Innovation, and UniversitiesRTI2018-095204-B-C21 Spain
CitationJournal: Nat Commun / Year: 2020
Title: Molecular architecture and activation of the insecticidal protein Vip3Aa from Bacillus thuringiensis.
Authors: Rafael Núñez-Ramírez / Juanjo Huesa / Yolanda Bel / Juan Ferré / Patricia Casino / Ernesto Arias-Palomo /
Abstract: Bacillus thuringiensis Vip3 (Vegetative Insecticidal Protein 3) toxins are widely used in biotech crops to control Lepidopteran pests. These proteins are produced as inactive protoxins that need to ...Bacillus thuringiensis Vip3 (Vegetative Insecticidal Protein 3) toxins are widely used in biotech crops to control Lepidopteran pests. These proteins are produced as inactive protoxins that need to be activated by midgut proteases to trigger cell death. However, little is known about their three-dimensional organization and activation mechanism at the molecular level. Here, we have determined the structures of the protoxin and the protease-activated state of Vip3Aa at 2.9 Å using cryo-electron microscopy. The reconstructions show that the protoxin assembles into a pyramid-shaped tetramer with the C-terminal domains exposed to the solvent and the N-terminal region folded into a spring-loaded apex that, after protease activation, drastically remodels into an extended needle by a mechanism akin to that of influenza haemagglutinin. These results provide the molecular basis for Vip3 activation and function, and serves as a strong foundation for the development of more efficient insecticidal proteins.
History
DepositionNov 14, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 12, 2020Provider: repository / Type: Initial release
Revision 1.1Aug 19, 2020Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Aug 26, 2020Group: Database references / Structure summary / Category: citation_author / struct_keywords
Item: _citation_author.identifier_ORCID / _struct_keywords.text

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Structure visualization

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Assembly

Deposited unit
A: Vegetative insecticidal protein
B: Vegetative insecticidal protein
C: Vegetative insecticidal protein
D: Vegetative insecticidal protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)355,0765
Polymers355,0514
Non-polymers241
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area21340 Å2
ΔGint-158 kcal/mol
Surface area121300 Å2
MethodPISA

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Components

#1: Protein
Vegetative insecticidal protein


Mass: 88762.805 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: The sample was activated by trypsin digestion / Source: (gene. exp.) Bacillus thuringiensis (bacteria) / Gene: vip3LB / Production host: Escherichia coli (E. coli) / References: UniProt: Q58XI2
#2: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Vip3Aa toxin / Type: COMPLEX / Details: The protein was activated by trypsin digestion / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.35 MDa / Experimental value: YES
Source (natural)Organism: Bacillus thuringiensis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 60 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18rc6_3830refinement
PHENIX1.18rc6_3830refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4GctfCTF correction
5RELION3CTF correction
10PHENIX1.17model refinement
11RELION3initial Euler assignment
12RELION3final Euler assignment
14RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 92303 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Details: The atomic coordinates were manually modeled de novo in the cryo-EM map using Coot, and then subjected to iterative rounds of real space refinement using Phenix
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 90.69 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002122056
ELECTRON MICROSCOPYf_angle_d0.442429856
ELECTRON MICROSCOPYf_chiral_restr0.04063436
ELECTRON MICROSCOPYf_plane_restr0.00243836
ELECTRON MICROSCOPYf_dihedral_angle_d11.20478216

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