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- PDB-9go9: Prepore state of alpha-Latrotoxin -

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Basic information

Entry
Database: PDB / ID: 9go9
TitlePrepore state of alpha-Latrotoxin
ComponentsAlpha-latrotoxin-Lt1a
KeywordsTOXIN / Black widow spider toxin / Pore forming neurotoxin / Ankyrin repeat / presynaptic receptor activation
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 (many copies) / Ankyrin repeat / 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-latrotoxin-Lt1a
Similarity search - Component
Biological speciesLatrodectus tredecimguttatus (black widow)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsKlink, B.U. / Gatsogiannis, C. / Kalyankumar, K.S.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation (DFG)SFB1348 (Project A15 to C.G.) Germany
Citation
Journal: Nat Commun / Year: 2024
Title: Structural basis of α-latrotoxin transition to a cation-selective pore.
Authors: B U Klink / A Alavizargar / K S Kalyankumar / M Chen / A Heuer / C Gatsogiannis /
Abstract: The potent neurotoxic venom of the black widow spider contains a cocktail of seven phylum-specific latrotoxins (LTXs), but only one, α-LTX, targets vertebrates. This 130 kDa toxin binds to ...The potent neurotoxic venom of the black widow spider contains a cocktail of seven phylum-specific latrotoxins (LTXs), but only one, α-LTX, targets vertebrates. This 130 kDa toxin binds to receptors at presynaptic nerve terminals and triggers a massive release of neurotransmitters. It is widely accepted that LTXs tetramerize and insert into the presynaptic membrane, thereby forming Ca-conductive pores, but the underlying mechanism remains poorly understood. LTXs are homologous and consist of an N-terminal region with three distinct domains, along with a C-terminal domain containing up to 22 consecutive ankyrin repeats. Here we report cryoEM structures of the vertebrate-specific α-LTX tetramer in its prepore and pore state. Our structures, in combination with AlphaFold2-based structural modeling and molecular dynamics simulations, reveal dramatic conformational changes in the N-terminal region of the complex. Four distinct helical bundles rearrange and together form a highly stable, 15 nm long, cation-impermeable coiled-coil stalk. This stalk, in turn, positions an N-terminal pair of helices within the membrane, thereby enabling the assembly of a cation-permeable channel. Taken together, these data give insight into a unique mechanism for membrane insertion and channel formation, characteristic of the LTX family, and provide the necessary framework for advancing novel therapeutics and biotechnological applications.
#1: Journal: BioRxiv / Year: 2024
Title: Molecular mechanism of alpha-latrotoxin action
Authors: Klink, B.U. / Alavizargar, A. / Kalyankumar, K.S. / Chen, M. / Heuer, A. / Gatsogiannis, C.
History
DepositionSep 5, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 16, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 6, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Alpha-latrotoxin-Lt1a
B: Alpha-latrotoxin-Lt1a
C: Alpha-latrotoxin-Lt1a
D: Alpha-latrotoxin-Lt1a


Theoretical massNumber of molelcules
Total (without water)628,1774
Polymers628,1774
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Alpha-latrotoxin-Lt1a / Alpha-LTX-Lt1a / Alpha-latrotoxin / Alpha-LTX


Mass: 157044.297 Da / Num. of mol.: 4 / Source method: isolated from a natural source
Details: alpha-latrotoxin activated via cleavage by Furin-like proteases
Source: (natural) Latrodectus tredecimguttatus (black widow)
Organ: venom gland / References: UniProt: P23631
Has protein modificationY

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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: tetrameric complex of alpha-latrotoxin / Type: COMPLEX
Details: The sample presents the activated form of latrotoxin derived by cleavage by Furin-like proteases. The complex is in the prepore conformation.
Entity ID: all / Source: NATURAL
Molecular weightValue: 0.526 MDa / Experimental value: NO
Source (natural)Organism: Latrodectus tredecimguttatus (black widow) / Organ: venom gland
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTRIS1
2150 mMsodium chlorideNaCl1
30.1 mMcalcium chlorideCaCl21
40.2 mMmagnesium chlorideMgCl21
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Monodisperse sample after gel filtration.
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: UltrAuFoil R2/2
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 286.15 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 215000 X / Nominal defocus max: 1700 nm / Nominal defocus min: 300 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 90215
Details: Images were collected in EER mode with 918-1225 frames per movie, from which 49-51 fractions were generated for motion correction. Micrographs were collected at different tilt angles in ten subsets of data.
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategoryDetails
1crYOLO1.8.3particle selectioncryolo was used to automatically select particles.
2EPU3.2.0.4776image acquisitionEPU was used to automatically collect EER movies
4RELION4.01CTF correction
7UCSF ChimeraX1.7.dev2023model fitting
9RELION4.01initial Euler assignment
10RELION4.01final Euler assignment
11RELION4.01classification
12RELION4.013D reconstruction
13Coot0.9.8.1model refinement
14PHENIX1.20.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2436143
Details: Particles were selected using crYOLO with a generalized picking model, followed by retraining with cleaned particle sets and repicking with a such derived optimized picking model.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 442105
Details: For the final reconstruction, Multibody-refinement, as implemented in Relion was performed, with a total of 8 independent bodies.
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 116 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: cross correlation 0.72
Details: An Alphafold2 prediction was initially fit into the reconstructiuon using ChimeraX and then manually refined using Coot and energy minimized using Phenix.
Atomic model buildingChain residue range: 21-1195
Details: The initial model was predicted from the monomeric, mature latrotoxin
Source name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 116.04 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002137660
ELECTRON MICROSCOPYf_angle_d0.520150988
ELECTRON MICROSCOPYf_chiral_restr0.03885812
ELECTRON MICROSCOPYf_plane_restr0.00436620
ELECTRON MICROSCOPYf_dihedral_angle_d3.82185040

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