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- PDB-7ptx: Alpha-latrocrustotoxin monomer -

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Basic information

Entry
Database: PDB / ID: 7ptx
TitleAlpha-latrocrustotoxin monomer
ComponentsAlpha-latrocrustotoxin-Lt1a
KeywordsTOXIN / Pore-forming toxin
Function / homology
Function and homology information


other organism cell membrane / host cell presynaptic membrane / exocytosis / toxin activity / extracellular region / membrane
Similarity search - Function
Ankyrin repeats (3 copies) / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats / Ankyrin repeat / Ankyrin repeat-containing domain superfamily
Similarity search - Domain/homology
Alpha-latrocrustotoxin-Lt1a
Similarity search - Component
Biological speciesLatrodectus tredecimguttatus (black widow)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.03 Å
AuthorsChen, M. / Gatsogiannis, C.
Funding support Japan, 3items
OrganizationGrant numberCountry
Alexander von Humboldt Foundation Japan
Max Planck Society Japan
Japan Society for the Promotion of Science (JSPS) Japan
CitationJournal: Nat Commun / Year: 2021
Title: Molecular architecture of black widow spider neurotoxins.
Authors: Minghao Chen / Daniel Blum / Lena Engelhard / Stefan Raunser / Richard Wagner / Christos Gatsogiannis /
Abstract: Latrotoxins (LaTXs) are presynaptic pore-forming neurotoxins found in the venom of Latrodectus spiders. The venom contains a toxic cocktail of seven LaTXs, with one of them targeting vertebrates (α- ...Latrotoxins (LaTXs) are presynaptic pore-forming neurotoxins found in the venom of Latrodectus spiders. The venom contains a toxic cocktail of seven LaTXs, with one of them targeting vertebrates (α-latrotoxin (α-LTX)), five specialized on insects (α, β, γ, δ, ε- latroinsectotoxins (LITs), and one on crustaceans (α-latrocrustatoxin (α-LCT)). LaTXs bind to specific receptors on the surface of neuronal cells, inducing the release of neurotransmitters either by directly stimulating exocytosis or by forming Ca-conductive tetrameric pores in the membrane. Despite extensive studies in the past decades, a high-resolution structure of a LaTX is not yet available and the precise mechanism of LaTX action remains unclear. Here, we report cryoEM structures of the α-LCT monomer and the δ-LIT dimer. The structures reveal that LaTXs are organized in four domains. A C-terminal domain of ankyrin-like repeats shields a central membrane insertion domain of six parallel α-helices. Both domains are flexibly linked via an N-terminal α-helical domain and a small β-sheet domain. A comparison between the structures suggests that oligomerization involves major conformational changes in LaTXs with longer C-terminal domains. Based on our data we propose a cyclic mechanism of oligomerization, taking place prior membrane insertion. Both recombinant α-LCT and δ-LIT form channels in artificial membrane bilayers, that are stabilized by Ca ions and allow calcium flux at negative membrane potentials. Our comparative analysis between α-LCT and δ-LIT provides first crucial insights towards understanding the molecular mechanism of the LaTX family.
History
DepositionSep 27, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 8, 2021Provider: repository / Type: Initial release
Revision 1.1Jul 17, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
A: Alpha-latrocrustotoxin-Lt1a


Theoretical massNumber of molelcules
Total (without water)140,0111
Polymers140,0111
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area49130 Å2

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Components

#1: Protein Alpha-latrocrustotoxin-Lt1a / Alpha-LCT-Lt1a / Alpha-latrocrustotoxin / Alpha-LCT / Crusta1


Mass: 140010.531 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Latrodectus tredecimguttatus (black widow)
Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9XZC0

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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: Alpha-latrocrustotoxin-Lt1a in soluble monomeric state
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.1398 MDa / Experimental value: NO
Source (natural)Organism: Latrodectus tredecimguttatus (black widow)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMtrisaminomethane hydrochlorideTris-HCl1
2150 mMsodium chlorideNaCl1
31 mMEthylenediaminetetraacetic acidEDTA1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 69.1 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1crYOLO1.4particle selection
4CTFFIND4.1.10CTF correction
7ISOLDE1.1.0model fitting
8Coot1model fitting
12RELION3.1classification
13SPHIRE1.33D reconstruction
14PHENIX1.18model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.03 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 376474 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL

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