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- EMDB-10493: Vip3Aa toxin structure -

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Basic information

Entry
Database: EMDB / ID: EMD-10493
TitleVip3Aa toxin structure
Map datasharpened cryo-EM map of the Vip3Aa toxin
Sample
  • Complex: Vip3Aa toxin
    • Protein or peptide: Vegetative insecticidal protein
  • Ligand: MAGNESIUM ION
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)
Methodsingle 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, 5 items
OrganizationGrant numberCountry
Spanish Ministry of Economy and CompetitivenessBFU2017-89143-P Spain
Spanish Ministry of Economy and CompetitivenessBFU2016-78606-P Spain
Spanish Ministry of Economy and CompetitivenessRYC-2015-19059 Spain
Spanish Ministry of Science, Innovation, and UniversitiesRTI2018-095204-B-C21 Spain
Spanish Ministry of Economy and CompetitivenessRYC-2014-16490 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, 2019-
Header (metadata) releaseAug 12, 2020-
Map releaseAug 12, 2020-
UpdateAug 26, 2020-
Current statusAug 26, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6tfk
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10493.png

Downloads & links

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Map

FileDownload / File: emd_10493.map.gz / Format: CCP4 / Size: 70.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened cryo-EM map of the Vip3Aa toxin
Voxel sizeX=Y=Z: 1.055 Å
Density
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.025
Minimum - Maximum-0.11826232 - 0.23407023
Average (Standard dev.)0.00031227065 (±0.004886643)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions264264264
Spacing264264264
CellA=B=C: 278.52 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0551.0551.055
M x/y/z264264264
origin x/y/z0.0000.0000.000
length x/y/z278.520278.520278.520
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS264264264
D min/max/mean-0.1180.2340.000

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Supplemental data

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Mask #1

Fileemd_10493_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened cryo-EM map of the Vip3Aa toxin

Fileemd_10493_additional_1.map
AnnotationUnsharpened cryo-EM map of the Vip3Aa toxin
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Focused classification map of the C-terminal domains of the toxin

Fileemd_10493_additional_2.map
AnnotationFocused classification map of the C-terminal domains of the toxin
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_10493_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_10493_half_map_2.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Vip3Aa toxin

EntireName: Vip3Aa toxin
Components
  • Complex: Vip3Aa toxin
    • Protein or peptide: Vegetative insecticidal protein
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Vip3Aa toxin

SupramoleculeName: Vip3Aa toxin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: The protein was activated by trypsin digestion
Source (natural)Organism: Bacillus thuringiensis (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightExperimental: 350 KDa

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Macromolecule #1: Vegetative insecticidal protein

MacromoleculeName: Vegetative insecticidal protein / type: protein_or_peptide / ID: 1 / Details: The sample was activated by trypsin digestion / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Bacillus thuringiensis (bacteria)
Molecular weightTheoretical: 88.762805 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNKNNTKLST RALPSFIDYF NGIYGFATGI KDIMNMIFKT DTGGDLTLDE ILKNQQLLND ISGKLDGVNG SLNDLIAQGN LNTELSKEI LKIANEQNQV LNDVNNKLDA INTMLRVYLP KLTSMLSDVM KQNYALSLQI EYLSKQLQEI SDKLDIINVN V LINSTLTE ...String:
MNKNNTKLST RALPSFIDYF NGIYGFATGI KDIMNMIFKT DTGGDLTLDE ILKNQQLLND ISGKLDGVNG SLNDLIAQGN LNTELSKEI LKIANEQNQV LNDVNNKLDA INTMLRVYLP KLTSMLSDVM KQNYALSLQI EYLSKQLQEI SDKLDIINVN V LINSTLTE ITPAYQRIKY VNEKFEELTF ATETSSKVKK DGSPADILDE LTELTELAKS VTKNDVDGFE FYLNTFHDVM VG NNLFGRS ALKTASELIT KENVKTSGSE VGNVYNFLIV LTALQAKAFL TLTTCRKLLG LADIDYTSIM NEHLNKEKEE FRV NILPTL SNTFSNPNYA KVKGSDEDAK MIVEAKPGHA LIGFEISNDS ITVLKVYEAK LKQNYQVDKD SLSEVIYGDM DKLL CPDQS EQIYYTNNIV FPNEYVITKI DFTKKMKTLR YEVTANFYDS STGEIDLNKK KVESSEAEYR TLSANDDGVY MPLGV ISET FLTPINGFGL QADENSRLIT LTCKSYLREL LLATDLSNKE TKLIVPPSGF ISNIVENGSI EEDNLEPWKA NNKNAY VDH TGGVNGTKAL YVHKDGGISQ FIGDKLKPKT EYVIQYTVKG KPSIHLKDEN TGYIHYEDTN NNLEDYQTIN KRFTTGT DL KGVYLILKSQ NGDEAWGDNF IILEISPSEK LLSPELINTN NWTSTGSTNI SGNTLTLYQG GRGILKQNLQ LDSFSTYR V YFSVSGDANV RIRNSREVLF EKRYMSGAKD VSEMFTTKFE KDNFYIELSQ GNNLYGGPIV HFYDVSIK

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Macromolecule #2: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

BufferpH: 8
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware: (Name: Gctf, RELION (ver. 3.0))
Startup modelType of model: OTHER
Details: Initial model was obtained using the SGD algorithm implemented in RELION
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 92303
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsThe 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
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-6tfk:
Vip3Aa toxin structure

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