[English] 日本語
Yorodumi
- PDB-8jlh: Cryo-EM structure of SV2A dimer in complex with BoNT/A2 Hc and le... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 8jlh
TitleCryo-EM structure of SV2A dimer in complex with BoNT/A2 Hc and levetiracetam
Components
  • Botulinum neurotoxin
  • Synaptic vesicle glycoprotein 2A
KeywordsTRANSPORT PROTEIN / Synaptic vesicle / epilepsy
Function / homology
Function and homology information


Toxicity of botulinum toxin type F (botF) / Toxicity of botulinum toxin type D (botD) / Toxicity of botulinum toxin type E (botE) / Toxicity of botulinum toxin type A (botA) / host cell presynaptic membrane / host cell cytoplasmic vesicle / presynaptic active zone / host cell cytosol / synaptic vesicle priming / protein transmembrane transporter activity ...Toxicity of botulinum toxin type F (botF) / Toxicity of botulinum toxin type D (botD) / Toxicity of botulinum toxin type E (botE) / Toxicity of botulinum toxin type A (botA) / host cell presynaptic membrane / host cell cytoplasmic vesicle / presynaptic active zone / host cell cytosol / synaptic vesicle priming / protein transmembrane transporter activity / transmembrane transporter activity / neuromuscular junction / GABA-ergic synapse / metalloendopeptidase activity / intracellular calcium ion homeostasis / synaptic vesicle / cell-cell junction / synaptic vesicle membrane / toxin activity / neuron projection / protein kinase binding / host cell plasma membrane / glutamatergic synapse / endoplasmic reticulum / proteolysis / extracellular region / zinc ion binding / plasma membrane
Similarity search - Function
: / SV2A/B/C luminal domain / Synaptic vesicle protein SV2 / Sugar transporter, conserved site / Major facilitator, sugar transporter-like / Sugar (and other) transporter / Clostridium neurotoxin, translocation / Clostridium neurotoxin, Translocation domain / Clostridium neurotoxin, translocation domain / Clostridium neurotoxin, receptor-binding C-terminal ...: / SV2A/B/C luminal domain / Synaptic vesicle protein SV2 / Sugar transporter, conserved site / Major facilitator, sugar transporter-like / Sugar (and other) transporter / Clostridium neurotoxin, translocation / Clostridium neurotoxin, Translocation domain / Clostridium neurotoxin, translocation domain / Clostridium neurotoxin, receptor-binding C-terminal / Clostridium neurotoxin, C-terminal receptor binding / Clostridial neurotoxin zinc protease / Botulinum/Tetanus toxin, catalytic chain / Clostridium neurotoxin, receptor binding N-terminal / Clostridium neurotoxin, N-terminal receptor binding / Kunitz inhibitor STI-like superfamily / Major facilitator superfamily / Major Facilitator Superfamily / Major facilitator superfamily domain / Major facilitator superfamily (MFS) profile. / MFS transporter superfamily / Concanavalin A-like lectin/glucanase domain superfamily
Similarity search - Domain/homology
: / Bontoxilysin-A / Synaptic vesicle glycoprotein 2A
Similarity search - Component
Biological speciesHomo sapiens (human)
Clostridium botulinum (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsYamagata, A.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP22H02564 Japan
CitationJournal: Nat Commun / Year: 2024
Title: Structural basis for antiepileptic drugs and botulinum neurotoxin recognition of SV2A.
Authors: Atsushi Yamagata / Kaori Ito / Takehiro Suzuki / Naoshi Dohmae / Tohru Terada / Mikako Shirouzu /
Abstract: More than one percent of people have epilepsy worldwide. Levetiracetam (LEV) is a successful new-generation antiepileptic drug (AED), and its derivative, brivaracetam (BRV), shows improved efficacy. ...More than one percent of people have epilepsy worldwide. Levetiracetam (LEV) is a successful new-generation antiepileptic drug (AED), and its derivative, brivaracetam (BRV), shows improved efficacy. Synaptic vesicle glycoprotein 2a (SV2A), a putative membrane transporter in the synaptic vesicles (SVs), has been identified as a target of LEV and BRV. SV2A also serves as a receptor for botulinum neurotoxin (BoNT), which is the most toxic protein and has paradoxically emerged as a potent reagent for therapeutic and cosmetic applications. Nevertheless, no structural analysis on AEDs and BoNT recognition by full-length SV2A has been available. Here we describe the cryo-electron microscopy structures of the full-length SV2A in complex with the BoNT receptor-binding domain, BoNT/A2 H and either LEV or BRV. The large fourth luminal domain of SV2A binds to BoNT/A2 H through protein-protein and protein-glycan interactions. LEV and BRV occupy the putative substrate-binding site in an outward-open conformation. A propyl group in BRV creates additional contacts with SV2A, explaining its higher binding affinity than that of LEV, which was further supported by label-free spectral shift assay. Numerous LEV derivatives have been developed as AEDs and positron emission tomography (PET) tracers for neuroimaging. Our work provides a structural framework for AEDs and BoNT recognition of SV2A and a blueprint for the rational design of additional AEDs and PET tracers.
History
DepositionJun 2, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 1, 2024Provider: repository / Type: Initial release
Revision 1.1May 8, 2024Group: Database references / Category: citation
Item: _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Oct 23, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

-
Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
A: Synaptic vesicle glycoprotein 2A
B: Botulinum neurotoxin
C: Synaptic vesicle glycoprotein 2A
D: Botulinum neurotoxin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)268,65711
Polymers266,4604
Non-polymers2,1967
Water00
1


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

-
Components

#1: Protein Synaptic vesicle glycoprotein 2A


Mass: 83774.008 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SV2A / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q7L0J3
#2: Protein Botulinum neurotoxin


Mass: 49456.191 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridium botulinum (bacteria) / Production host: Escherichia coli (E. coli) / References: UniProt: D2KCK3
#3: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta- ...2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 570.542 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4[LFucpa1-6]DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1221m-1a_1-5]/1-1-2/a4-b1_a6-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}[(6+1)][a-L-Fucp]{}}LINUCSPDB-CARE
#4: Chemical ChemComp-UKX / (2S)-2-(2-oxidanylidenepyrrolidin-1-yl)butanamide / levetiracetam


Mass: 170.209 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H14N2O2 / Feature type: SUBJECT OF INVESTIGATION
#5: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestY
Has protein modificationY

-
Experimental details

-
Experiment

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

-
Sample preparation

ComponentName: SV2A dimer in complex with BoNT/A2 Hc domain and anti-epileptic drug, levetiracetam
Type: COMPLEX / Entity ID: #2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Clostridium botulinum (bacteria)
Buffer solutionpH: 7.5
Details: 20 mM Hepes (pH7.5), 150 mM NaCl, 0.001% LMNG, 0.0002% cholesterol hemisuccinate
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 297 K

-
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 defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Image recordingElectron dose: 50.8 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6518

-
Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
4cryoSPARCCTF correction
7Cootmodel fitting
9REFMACmodel refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6409122
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 425512 / Symmetry type: POINT
RefinementResolution: 2.9→159.36 Å / Cor.coef. Fo:Fc: 0.856 / WRfactor Rwork: 0.374 / SU B: 10.939 / SU ML: 0.199 / Average fsc free: 0 / Average fsc overall: 0.8139 / Average fsc work: 0.8139 / Cross valid method: NONE / ESU R: 0.306
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rwork0.3745 209277 -
all0.374 --
Rfree--0 %
obs--100 %
Solvent computationSolvent model: NONE
Displacement parametersBiso mean: 158.273 Å2
Refinement stepCycle: 1 / Total: 12610
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00312897
ELECTRON MICROSCOPYf_angle_d0.57317434
ELECTRON MICROSCOPYf_dihedral_angle_d4.7621758
ELECTRON MICROSCOPYf_chiral_restr0.0441916
ELECTRON MICROSCOPYf_plane_restr0.0042203
LS refinement shell

Refine-ID: ELECTRON MICROSCOPY / Num. reflection Rfree: _ / Total num. of bins used: 20 / % reflection obs: 100 %

Resolution (Å)Rfactor RworkNum. reflection RworkRfactor allNum. reflection allFsc workWRfactor Rwork
2.9-2.9751.491154821.491154820.4551.491
2.975-3.0570.928150710.928150710.5730.928
3.057-3.1450.741147240.741147240.6790.741
3.145-3.2420.595141700.595141700.7550.595
3.242-3.3480.471138320.471138320.8130.471
3.348-3.4660.356133920.356133920.8590.356
3.466-3.5970.285129220.285129220.8910.285
3.597-3.7430.267123340.267123340.9070.267
3.743-3.910.273119480.273119480.9130.273
3.91-4.1010.297113490.297113490.9170.297
4.101-4.3220.339108090.339108090.9190.339
4.322-4.5840.363102750.363102750.9290.363
4.584-4.90.35596080.35596080.940.355
4.9-5.2930.32388970.32388970.9220.323
5.293-5.7970.29282450.29282450.8930.292
5.797-6.480.28974640.28974640.8820.289
6.48-7.4810.3165340.3165340.8650.31
7.481-9.1570.31555840.31555840.8620.315
9.157-12.9290.28642820.28642820.8970.286
12.929-159.360.68423580.68423580.9570.684

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more