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- EMDB-51494: Prepore state of alpha-Latrotoxin (hybrid map) -

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Basic information

Entry
Database: EMDB / ID: EMD-51494
TitlePrepore state of alpha-Latrotoxin (hybrid map)
Map dataPhenix Hybrid map calculated from 20 individual maps from consensus and focussed refinements
Sample
  • Complex: tetrameric complex of alpha-latrotoxin
    • Protein or peptide: Alpha-latrotoxin-Lt1a
KeywordsBlack widow spider toxin / Pore forming neurotoxin / Ankyrin repeat / presynaptic receptor activation / 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 (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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsKlink BU / Gatsogiannis C / Kalyankumar KS
Funding support Germany, 1 items
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 BU / Alavizargar A / Kalyankumar KS / Chen M / Heuer A / Gatsogiannis C
History
DepositionSep 5, 2024-
Header (metadata) releaseOct 16, 2024-
Map releaseOct 16, 2024-
UpdateNov 6, 2024-
Current statusNov 6, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_51494.png

Downloads & links

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Map

FileDownload / File: emd_51494.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPhenix Hybrid map calculated from 20 individual maps from consensus and focussed refinements
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.16 Å/pix.
x 280 pix.
= 324.8 Å		1.16 Å/pix.
x 280 pix.
= 324.8 Å		1.16 Å/pix.
x 280 pix.
= 324.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.16 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.2
Minimum - Maximum-38.633366000000002 - 56.782620000000001
Average (Standard dev.)-0.090157665 (±1.2716445)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 324.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : tetrameric complex of alpha-latrotoxin

EntireName: tetrameric complex of alpha-latrotoxin
Components
  • Complex: tetrameric complex of alpha-latrotoxin
    • Protein or peptide: Alpha-latrotoxin-Lt1a

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Supramolecule #1: tetrameric complex of alpha-latrotoxin

SupramoleculeName: tetrameric complex of alpha-latrotoxin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: The sample presents the activated form of latrotoxin derived by cleavage by Furin-like proteases. The complex is in the prepore conformation.
Source (natural)Organism: Latrodectus tredecimguttatus (black widow) / Organ: venom gland
Molecular weightTheoretical: 526 KDa

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Macromolecule #1: Alpha-latrotoxin-Lt1a

MacromoleculeName: Alpha-latrotoxin-Lt1a / type: protein_or_peptide / ID: 1
Details: alpha-latrotoxin activated via cleavage by Furin-like proteases
Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Latrodectus tredecimguttatus (black widow) / Organ: venom gland
Molecular weightTheoretical: 157.044297 KDa
SequenceString: MISVGEIMER ANHSLVRMRR EGEDLTLEEK AEICSELELQ QKYVDIASNI IGDLSSLPIA GKIAGTIAAA AMTATHVASG RLDIEQTLL GCSDLPFDQI KEVLENRFNE IDRKLDSHSA ALEEITKLVE KSISVVEKTR KQMNKRFDEV MKSIQDAKVS P IISKINNF ...String:
MISVGEIMER ANHSLVRMRR EGEDLTLEEK AEICSELELQ QKYVDIASNI IGDLSSLPIA GKIAGTIAAA AMTATHVASG RLDIEQTLL GCSDLPFDQI KEVLENRFNE IDRKLDSHSA ALEEITKLVE KSISVVEKTR KQMNKRFDEV MKSIQDAKVS P IISKINNF ARYFDTEKER IRGLKLNDYI LKLEEPNGIL LHFKESRTPT DDSLQAPLFS IIEEGYAVPK SIDDELAFKV LY ALLYGTQ TYVSVMFFLL EQYSFLANHY YEKGYLEKYD EYFNSLNNVF LDFKSSLVGT GTSNNEGLLD RVLQVLMTVK NSE FLGLEK NGVDEMLNEK INLFNKIKEE IEGKQKMTLS ETPENFAQIS FDKDITTPIG DWRDGREVRY AVQYASETLF SKIS HWSDP VSVREKACPT LRMPVDQTRR NVLVFRKFDS SKPQLVGEIT PYLSNFIDID RDLYNAASNP DSAVGFKEFT KLNYD GANI RATFDHGRTV FHAAAKSGND KIMFGLTFLA KSTELNQPDK KGYTPIHVAA DSGNAGIVNL LIQRGVSINS KTYHFL QTP LHLAAQRGFV TTFQRLMESP EININERDKD GFTPLHYAIR GGERILEAFL NQISIDVNAK SNTGLTPFHL AIIKNDW PV ASTLLGSKKV DINAVDENNI TALHYAAILG YLETTKQLIN LKEINANVVS SPGLLSALHY AILYKHDDVA SFLMRSSN V NVNLKALGGI TPLHLAVIQG RKQILSLMFD IGVNIEQKTD EKYTPLHLAA MSKYPELIQI LLDQGSNFEA KTNSGATPL HLATFKGKSQ AALILLNNEV NWRDTDENGQ MPIHGAAMTG LLDVAQAIIS IDATVVDIED KNSDTPLNLA AQNSHIDVIK YFIDQGADI NTRNKKGLAP LLAFSKKGNL DMVKYLFDKN ANVYIADNDG MNFFYYAVQN GHLNIVKYAM SEKDKFEWSN T DNNRRDEC PNEECAISHF AVCDAVQFDR IEIVKYFVGT LGNFAICGPL HQAARYGHLD IVKYLVEEEF LSVDGSKTDT PL CYASENG HFTVVQYLVS NGAKVNHDCG NGMTAIDKAI TKNHLQVVQF LAANGVDFRR KNSRGTTPFL TAVAENALHI AEY LIREKR QDININEQNV DKDTALHLAV YYKNLQMIKL LIKYGIDVTI RNAYDKTALD IAIDAKFSNI VEYLKTKSGK FRRE YKSSY GERSLLQTNQ ISNFIDRKNI EHDHPLFINA DNESSELFSK TASNIDVIGT LLLIDVLIRY FSKQGYISKE SDSAS DGIT QAAALSITEK FEDVLNSLHN ESAKEQVDLA EVHGKVYAAL KSGRNSQIHQ ILCSSLNSIS TLKPEDMEKL ESVIMN SHS SVSLPEVTDS ANEAYGETLH LFGESCLHSD GILTKKLM

UniProtKB: Alpha-latrotoxin-Lt1a

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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

Concentration0.5 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
25.0 mMTRIS
150.0 mMsodium chlorideNaCl
0.1 mMcalcium chlorideCaCl2
0.2 mMmagnesium chlorideMgCl2
GridModel: UltrAuFoil R2/2 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 286.15 K / Instrument: FEI VITROBOT MARK II
DetailsMonodisperse sample after gel filtration.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 3 / Number real images: 90215 / Average electron dose: 50.0 e/Å2
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.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.7 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 215000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber 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.
Startup modelType of model: NONE
Details: The initial model was generated in RELION using the "3D initial model" function.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.01)
Details: For the final reconstruction, Multibody-refinement, as implemented in Relion was performed, with a total of 8 independent bodies.
Number images used: 442105
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.01)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4.01)
Final 3D classificationNumber classes: 4 / Avg.num./class: 442105 / Software - Name: RELION (ver. 4.01)

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Atomic model buiding 1

Initial modelChain - Residue range: 21-1195 / Chain - Source name: AlphaFold / Chain - Initial model type: in silico model
Details: The initial model was predicted from the monomeric, mature latrotoxin
DetailsAn Alphafold2 prediction was initially fit into the reconstructiuon using ChimeraX and then manually refined using Coot and energy minimized using Phenix.
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 116 / Target criteria: cross correlation 0.72
Output model

PDB-9go9:
Prepore state of alpha-Latrotoxin

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