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- PDB-2n1t: Dynamic binding mode of a synaptotagmin-1-SNARE complex in solution -

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Entry
Database: PDB / ID: 2n1t
TitleDynamic binding mode of a synaptotagmin-1-SNARE complex in solution
Components
  • (Synaptosomal-associated protein 25) x 2
  • Synaptotagmin-1
  • Syntaxin-1A
  • Vesicle-associated membrane protein 2
KeywordsEXOCYTOSIS / Synaptotagmin-1 / C2B domain / Syntaxin-1A / Synaptobrevin-2 / SNAP-25 / SNAP-25A / SNARE complex
Function / homology
Function and homology information


Toxicity of botulinum toxin type C (botC) / exocytic insertion of neurotransmitter receptor to postsynaptic membrane / Toxicity of botulinum toxin type G (botG) / clathrin-sculpted acetylcholine transport vesicle membrane / trans-Golgi Network Vesicle Budding / clathrin-sculpted glutamate transport vesicle membrane / regulation of delayed rectifier potassium channel activity / neurotransmitter uptake / synchronous neurotransmitter secretion / fast, calcium ion-dependent exocytosis of neurotransmitter ...Toxicity of botulinum toxin type C (botC) / exocytic insertion of neurotransmitter receptor to postsynaptic membrane / Toxicity of botulinum toxin type G (botG) / clathrin-sculpted acetylcholine transport vesicle membrane / trans-Golgi Network Vesicle Budding / clathrin-sculpted glutamate transport vesicle membrane / regulation of delayed rectifier potassium channel activity / neurotransmitter uptake / synchronous neurotransmitter secretion / fast, calcium ion-dependent exocytosis of neurotransmitter / syntaxin-3 binding / Toxicity of botulinum toxin type E (botE) / spontaneous neurotransmitter secretion / regulation of regulated secretory pathway / Toxicity of botulinum toxin type B (botB) / clathrin-sculpted gamma-aminobutyric acid transport vesicle membrane / calcium-dependent activation of synaptic vesicle fusion / Lysosome Vesicle Biogenesis / myosin head/neck binding / positive regulation of calcium ion-dependent exocytosis of neurotransmitter / chromaffin granule membrane / GABA synthesis, release, reuptake and degradation / zymogen granule membrane / storage vacuole / synaptic vesicle fusion to presynaptic active zone membrane / Toxicity of botulinum toxin type A (botA) / Acetylcholine Neurotransmitter Release Cycle / Other interleukin signaling / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin II complex / synaptobrevin 2-SNAP-25-syntaxin-1a complex / presynaptic dense core vesicle exocytosis / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin I complex / clathrin-sculpted monoamine transport vesicle membrane / Glutamate Neurotransmitter Release Cycle / calcium ion sensor activity / Norepinephrine Neurotransmitter Release Cycle / regulation of calcium ion-dependent exocytosis / Acetylcholine Neurotransmitter Release Cycle / Serotonin Neurotransmitter Release Cycle / Serotonin Neurotransmitter Release Cycle / GABA synthesis, release, reuptake and degradation / positive regulation of norepinephrine secretion / positive regulation of catecholamine secretion / Dopamine Neurotransmitter Release Cycle / synaptic vesicle docking / eosinophil degranulation / Golgi Associated Vesicle Biogenesis / regulation of synaptic vesicle priming / regulated exocytosis / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / vesicle-mediated transport in synapse / positive regulation of intracellular protein transport / exocytic vesicle / Norepinephrine Neurotransmitter Release Cycle / Dopamine Neurotransmitter Release Cycle / positive regulation of calcium ion-dependent exocytosis / vesicle organization / protein heterooligomerization / ribbon synapse / positive regulation of dendrite extension / vesicle docking / regulation of vesicle-mediated transport / Cargo recognition for clathrin-mediated endocytosis / regulation of exocytosis / secretion by cell / chloride channel inhibitor activity / Glutamate Neurotransmitter Release Cycle / Clathrin-mediated endocytosis / positive regulation of dopamine secretion / SNARE complex / SNAP receptor activity / calcium-ion regulated exocytosis / vesicle fusion / actomyosin / hormone secretion / LGI-ADAM interactions / dense core granule / calcium-dependent phospholipid binding / neuron projection terminus / membraneless organelle assembly / Golgi to plasma membrane protein transport / ATP-dependent protein binding / neurotransmitter secretion / protein localization to membrane / clathrin-coated vesicle / syntaxin binding / presynaptic active zone / Neurexins and neuroligins / syntaxin-1 binding / insulin secretion / regulation of synaptic vesicle recycling / Sensory processing of sound by inner hair cells of the cochlea / Other interleukin signaling / low-density lipoprotein particle receptor binding / clathrin binding / phosphatidylserine binding / neurotransmitter transport / SNARE complex assembly / positive regulation of neurotransmitter secretion / myosin binding
Similarity search - Function
Single alpha-helices involved in coiled-coils or other helix-helix interfaces - #110 / Synaptobrevin/Vesicle-associated membrane protein / Synaptobrevin signature. / Synaptotagmin / Synaptosomal-associated protein 25 / SNAP-25 domain / Synaptobrevin-like / SNAP-25 family / Synaptobrevin / v-SNARE, coiled-coil homology domain ...Single alpha-helices involved in coiled-coils or other helix-helix interfaces - #110 / Synaptobrevin/Vesicle-associated membrane protein / Synaptobrevin signature. / Synaptotagmin / Synaptosomal-associated protein 25 / SNAP-25 domain / Synaptobrevin-like / SNAP-25 family / Synaptobrevin / v-SNARE, coiled-coil homology domain / v-SNARE coiled-coil homology domain profile. / Syntaxin / Syntaxin N-terminal domain / Syntaxin, N-terminal domain / SNARE domain / Syntaxin / Syntaxin/epimorphin, conserved site / Syntaxin / epimorphin family signature. / SNARE / Helical region found in SNAREs / t-SNARE coiled-coil homology domain profile. / Target SNARE coiled-coil homology domain / C2 domain / Protein kinase C conserved region 2 (CalB) / C2 domain / C2 domain / C2 domain profile. / C2 domain superfamily / Single alpha-helices involved in coiled-coils or other helix-helix interfaces / Up-down Bundle / Immunoglobulin-like / Sandwich / Mainly Beta / Mainly Alpha
Similarity search - Domain/homology
Synaptotagmin-1 / Syntaxin-1A / Synaptosomal-associated protein 25 / Vesicle-associated membrane protein 2
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Homo sapiens (human)
MethodSOLUTION NMR / molecular dynamics
AuthorsBrewer, K. / Bacaj, T. / Cavalli, A. / Camilloni, C. / Swarbrick, J. / Liu, J. / Zhou, A. / Zhou, P. / Barlow, N. / Xu, J. ...Brewer, K. / Bacaj, T. / Cavalli, A. / Camilloni, C. / Swarbrick, J. / Liu, J. / Zhou, A. / Zhou, P. / Barlow, N. / Xu, J. / Seven, A. / Prinslow, E. / Voleti, R. / Haussinger, D. / Bonvin, A. / Tomchick, D. / Vendruscolo, M. / Graham, B. / Sudhof, T. / Rizo, J.
CitationJournal: Nat.Struct.Mol.Biol. / Year: 2015
Title: Dynamic binding mode of a Synaptotagmin-1-SNARE complex in solution.
Authors: Brewer, K.D. / Bacaj, T. / Cavalli, A. / Camilloni, C. / Swarbrick, J.D. / Liu, J. / Zhou, A. / Zhou, P. / Barlow, N. / Xu, J. / Seven, A.B. / Prinslow, E.A. / Voleti, R. / Haussinger, D. / ...Authors: Brewer, K.D. / Bacaj, T. / Cavalli, A. / Camilloni, C. / Swarbrick, J.D. / Liu, J. / Zhou, A. / Zhou, P. / Barlow, N. / Xu, J. / Seven, A.B. / Prinslow, E.A. / Voleti, R. / Haussinger, D. / Bonvin, A.M. / Tomchick, D.R. / Vendruscolo, M. / Graham, B. / Sudhof, T.C. / Rizo, J.
History
DepositionApr 21, 2015Deposition site: BMRB / Processing site: RCSB
Revision 1.0Jun 3, 2015Provider: repository / Type: Initial release
Revision 1.1Jun 17, 2015Group: Database references
Revision 1.2Jul 22, 2015Group: Database references
Revision 1.3Aug 18, 2021Group: Data collection / Database references ...Data collection / Database references / Experimental preparation / Refinement description
Category: database_2 / pdbx_nmr_exptl ...database_2 / pdbx_nmr_exptl / pdbx_nmr_exptl_sample / pdbx_nmr_refine / pdbx_nmr_sample_details / pdbx_nmr_software / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_nmr_refine.software_ordinal / _pdbx_nmr_sample_details.contents / _pdbx_nmr_sample_details.label / _pdbx_nmr_sample_details.type / _pdbx_nmr_software.name / _struct_ref_seq_dif.details
Revision 1.4May 15, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2 / Item: _database_2.pdbx_DOI

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Vesicle-associated membrane protein 2
B: Syntaxin-1A
C: Synaptosomal-associated protein 25
D: Synaptosomal-associated protein 25
E: Synaptotagmin-1


Theoretical massNumber of molelcules
Total (without water)51,4285
Polymers51,4285
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area15170 Å2
ΔGint-97 kcal/mol
Surface area23800 Å2
MethodPISA
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)5 / 10000Contribution to fit PCS data
RepresentativeModel #1all these conformers contribute to this dynamics ensemble

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Components

#1: Protein Vesicle-associated membrane protein 2 / VAMP-2 / Synaptobrevin-2


Mass: 8045.023 Da / Num. of mol.: 1 / Fragment: UNP residues 25-93
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Syb2, Vamp2 / Plasmid: pGEX-KT / Production host: Escherichia coli (E. coli) / References: UniProt: P63045
#2: Protein Syntaxin-1A / Neuron-specific antigen HPC-1 / Synaptotagmin-associated 35 kDa protein / P35A


Mass: 8429.528 Da / Num. of mol.: 1 / Fragment: UNP residues 188-259
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Sap, Stx1a / Plasmid: pGEX-KT / Production host: Escherichia coli (E. coli) / References: UniProt: P32851
#3: Protein Synaptosomal-associated protein 25 / SNAP-25 / SNAP-25A / Super protein / SUP / Synaptosomal-associated 25 kDa protein


Mass: 9030.114 Da / Num. of mol.: 1 / Fragment: N-terminal domain (UNP residues 7-83)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SNAP, SNAP25, SNAP-25A / Plasmid: pGEX-KT / Production host: Escherichia coli (E. coli) / References: UniProt: P60880
#4: Protein Synaptosomal-associated protein 25 / SNAP-25 / SNAP-25A / Super protein / SUP / Synaptosomal-associated 25 kDa protein


Mass: 8458.420 Da / Num. of mol.: 1 / Fragment: C-terminal domain (UNP residues 131-204)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SNAP, SNAP25, SNAP-25A / Plasmid: pGEX-KT / Production host: Escherichia coli (E. coli) / References: UniProt: P60880
#5: Protein Synaptotagmin-1 / Synaptotagmin I / SytI / p65


Mass: 17465.236 Da / Num. of mol.: 1 / Fragment: C2B domain (UNP residues 272-419)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SVP65, SYT, SYT1 / Plasmid: pGEX-KT / Production host: Escherichia coli (E. coli) / References: UniProt: P21579
Compound detailsRESIDUE ASP 41 OF SNAP-25 N-TERMINAL DOMAIN (CHAIN C) WAS MUTATED TO CYS, AND LABELED WITH DY-C2 ...RESIDUE ASP 41 OF SNAP-25 N-TERMINAL DOMAIN (CHAIN C) WAS MUTATED TO CYS, AND LABELED WITH DY-C2 TAG IN SOME SAMPLES.
Sequence detailsTHE SEQUENCES OF CHAIN C AND D MATCH ISOFORM 2 SEQUENCE WITH UNIPROT IDENTIFIER P60880-2.

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
Details: This deposition includes five conformers of the synaptotagmin-1 C2B domain SNARE complex. This complex is highly dynamic under the solution conditions used in the measurement of ...Details: This deposition includes five conformers of the synaptotagmin-1 C2B domain SNARE complex. This complex is highly dynamic under the solution conditions used in the measurement of pseudocontact shifts by NMR spectroscopy that provided the structural information for this analysis. Because of this highly dynamic nature, no single structure can be considered as 'the structure' of this complex under our conditions. The five conformers deposited must be considered as just a few of the many conformers that form the ensemble but illustrate the types of interactions between the synaptotagmin-1 C2B domain and the SNARE complex that mediate this dynamic binding mode.
NMR experiment
Conditions-IDExperiment-IDSolution-IDType
1112D 1H-15N HSQC
1212D 1H-13C HMQC

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Sample preparation

DetailsType: solution
Contents: 30 uM [U-100% 13C; U-100% 15N] protein, 25 mM Tris-HCl, 125 mM KSCN, 1 mM CaCl2, 90% H2O/10% D2O
Label: sample_1 / Solvent system: 90% H2O/10% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
30 uMprotein[U-100% 13C; U-100% 15N]1
25 mMTris-HClnatural abundance1
125 mMKSCNnatural abundance1
1 mMCaCl2natural abundance1
Sample conditionspH: 7.4 / Pressure: ambient / Temperature: 298 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Varian INOVAVarianINOVA8001
Varian INOVAVarianINOVA6002

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Processing

NMR software
NameVersionDeveloperClassification
NMRView8.0.a15Johnson, One Moon Scientificdata analysis
Numbat0.991Christophe Schmitz and Thomas Huberdata analysis
NAMD2.7Klaus Schultenrefinement
GROMACSvan Gunsteren and Berendsenrefinement
PLUMED2Giovanni Bussirefinement
ALMOSTAndrea Cavallirefinement
RefinementMethod: molecular dynamics / Software ordinal: 3
Details: MOLECULAR DYNAMICS SIMULATIONS WERE USED TO GENERATE STRUCTURES OF THE SYNAPTOTAGMIN-1 C2B DOMAIN-SNARE COMPLEX THAT COULD BE EVALUATED BASED ON HOW WELL THEY FIT THE MEASURED PSEUDOCONTACT ...Details: MOLECULAR DYNAMICS SIMULATIONS WERE USED TO GENERATE STRUCTURES OF THE SYNAPTOTAGMIN-1 C2B DOMAIN-SNARE COMPLEX THAT COULD BE EVALUATED BASED ON HOW WELL THEY FIT THE MEASURED PSEUDOCONTACT SHIFTS. SINCE IT WAS CLEAR FROM THE ANALYSIS THAT THE COMPLEX IS HIGHLY DYNAMICS AND NO SINGLE STRUCTURE CAN FIT ALL THE DATA, THE STRUCTURES FROM THE SIMULATIONS WERE USED TO TRY TO FIT THE MEASURED PSEUDOCONTACT SHIFTS AS ENSEMBLED-AVERAGED VALUES. THE STRUCTURES IN THE DEPOSITION ARE AMONG THOSE THAT CONTRIBUTED TO THE BEST ENSEMBLE-AVERAGE FITS.
NMR representativeSelection criteria: all these conformers contribute to this dynamics ensemble
NMR ensembleConformer selection criteria: Contribution to fit PCS data / Conformers calculated total number: 10000 / Conformers submitted total number: 5

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