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- EMDB-9231: Cryo-EM structure of membrane-bound Synaptotagmin 1 C2AB domains ... -

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Basic information

Entry
Database: EMDB / ID: EMD-9231
TitleCryo-EM structure of membrane-bound Synaptotagmin 1 C2AB domains in a complex with the SNAREpin assembly in presence of Mg2+
Map dataCryo-EM map of Syt1-C2AB-SNAREpin complexes on the surface of negatively charged lipid nanotube flipped along z-axis. 10.4A resolution.
Sample
  • Complex: Synaptotagmin 1 C2AB in a complex with the SNAREpin assembly bound to the negatively charged phospholipid nanotube in presence of Mg2+.
    • Protein or peptide: Synaptotagmin 1
    • Protein or peptide: Vesicle-associated membrane protein 2
    • Protein or peptide: Syntaxin-1A
    • Protein or peptide: Synaptosomal-associated protein 25
    • Protein or peptide: Synaptosomal-associated protein 25
Function / homology
Function and homology information


trans-Golgi Network Vesicle Budding / BLOC-1 complex / regulation of delayed rectifier potassium channel activity / myosin head/neck binding / synchronous neurotransmitter secretion / fast, calcium ion-dependent exocytosis of neurotransmitter / positive regulation of calcium ion-dependent exocytosis of neurotransmitter / syntaxin-3 binding / exocytic insertion of neurotransmitter receptor to postsynaptic membrane / Other interleukin signaling ...trans-Golgi Network Vesicle Budding / BLOC-1 complex / regulation of delayed rectifier potassium channel activity / myosin head/neck binding / synchronous neurotransmitter secretion / fast, calcium ion-dependent exocytosis of neurotransmitter / positive regulation of calcium ion-dependent exocytosis of neurotransmitter / syntaxin-3 binding / exocytic insertion of neurotransmitter receptor to postsynaptic membrane / Other interleukin signaling / calcium-dependent activation of synaptic vesicle fusion / calcium ion sensor activity / regulation of regulated secretory pathway / extrinsic component of presynaptic membrane / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin II complex / synaptobrevin 2-SNAP-25-syntaxin-1a complex / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin I complex / spontaneous neurotransmitter secretion / Lysosome Vesicle Biogenesis / synaptic vesicle fusion to presynaptic active zone membrane / regulation of synaptic vesicle priming / Glutamate Neurotransmitter Release Cycle / positive regulation of norepinephrine secretion / Norepinephrine Neurotransmitter Release Cycle / Acetylcholine Neurotransmitter Release Cycle / positive regulation of catecholamine secretion / zymogen granule membrane / Serotonin Neurotransmitter Release Cycle / dense core granule / GABA synthesis, release, reuptake and degradation / positive regulation of vesicle fusion / chromaffin granule membrane / Dopamine Neurotransmitter Release Cycle / Golgi Associated Vesicle Biogenesis / regulated exocytosis / presynaptic dense core vesicle exocytosis / ribbon synapse / synaptic vesicle docking / regulation of establishment of protein localization / storage vacuole / response to gravity / calcium ion-regulated exocytosis of neurotransmitter / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / eosinophil degranulation / vesicle fusion / regulation of calcium ion-dependent exocytosis / vesicle docking / positive regulation of calcium ion-dependent exocytosis / exocytic vesicle / chloride channel inhibitor activity / secretion by cell / SNARE complex / SNAP receptor activity / positive regulation of dopamine secretion / protein heterooligomerization / Cargo recognition for clathrin-mediated endocytosis / regulation of vesicle-mediated transport / Clathrin-mediated endocytosis / LGI-ADAM interactions / hormone secretion / calcium-ion regulated exocytosis / actomyosin / positive regulation of intracellular protein transport / positive regulation of dendrite extension / Golgi to plasma membrane protein transport / regulation of exocytosis / positive regulation of hormone secretion / neurotransmitter secretion / neurotransmitter receptor internalization / calcium-dependent phospholipid binding / protein localization to membrane / ATP-dependent protein binding / neuron projection terminus / neurotransmitter transport / regulation of synaptic vesicle recycling / insulin secretion / syntaxin-1 binding / SNARE complex assembly / positive regulation of neurotransmitter secretion / syntaxin binding / clathrin-coated vesicle / Neutrophil degranulation / synaptic vesicle priming / regulation of synapse assembly / regulation of neuron projection development / endosomal transport / regulation of synaptic vesicle exocytosis / low-density lipoprotein particle receptor binding / clathrin binding / myosin binding / regulation of dopamine secretion / phosphatidylserine binding / exocytosis / voltage-gated potassium channel activity / positive regulation of exocytosis / synaptic vesicle exocytosis / modulation of excitatory postsynaptic potential / associative learning / positive regulation of excitatory postsynaptic potential / protein sumoylation
Similarity search - Function
Synaptobrevin/Vesicle-associated membrane protein / Synaptosomal-associated protein 25 / SNAP-25 domain / SNAP-25 family / Synaptobrevin signature. / Syntaxin / Syntaxin N-terminal domain / Syntaxin, N-terminal domain / Syntaxin / SNARE domain ...Synaptobrevin/Vesicle-associated membrane protein / Synaptosomal-associated protein 25 / SNAP-25 domain / SNAP-25 family / Synaptobrevin signature. / Syntaxin / Syntaxin N-terminal domain / Syntaxin, N-terminal domain / Syntaxin / SNARE domain / Synaptobrevin-like / Syntaxin/epimorphin, conserved site / Synaptobrevin / Syntaxin / epimorphin family signature. / v-SNARE, coiled-coil homology domain / v-SNARE coiled-coil homology domain profile. / SNARE / Synaptotagmin / 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 profile. / C2 domain superfamily
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)
Methodhelical reconstruction / cryo EM / Resolution: 10.4 Å
AuthorsGrushin K / Wang J / Coleman J / Rothman J / Sindelar C / Krishnakumar S
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney DiseaseDK027044 United States
CitationJournal: Nat Commun / Year: 2019
Title: Structural basis for the clamping and Ca activation of SNARE-mediated fusion by synaptotagmin.
Authors: Kirill Grushin / Jing Wang / Jeff Coleman / James E Rothman / Charles V Sindelar / Shyam S Krishnakumar /
Abstract: Synapotagmin-1 (Syt1) interacts with both SNARE proteins and lipid membranes to synchronize neurotransmitter release to calcium (Ca) influx. Here we report the cryo-electron microscopy structure of ...Synapotagmin-1 (Syt1) interacts with both SNARE proteins and lipid membranes to synchronize neurotransmitter release to calcium (Ca) influx. Here we report the cryo-electron microscopy structure of the Syt1-SNARE complex on anionic-lipid containing membranes. Under resting conditions, the Syt1 C2 domains bind the membrane with a magnesium (Mg)-mediated partial insertion of the aliphatic loops, alongside weak interactions with the anionic lipid headgroups. The C2B domain concurrently interacts the SNARE bundle via the 'primary' interface and is positioned between the SNAREpins and the membrane. In this configuration, Syt1 is projected to sterically delay the complete assembly of the associated SNAREpins and thus, contribute to clamping fusion. This Syt1-SNARE organization is disrupted upon Ca-influx as Syt1 reorients into the membrane, likely displacing the attached SNAREpins and reversing the fusion clamp. We thus conclude that the cation (Mg/Ca) dependent membrane interaction is a key determinant of the dual clamp/activator function of Synaptotagmin-1.
History
DepositionOct 17, 2018-
Header (metadata) releaseNov 14, 2018-
Map releaseApr 24, 2019-
UpdateNov 6, 2019-
Current statusNov 6, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0134
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0134
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6mti
  • Surface level: 0.0134
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6mti
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9231.png

Downloads & links

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Map

FileDownload / File: emd_9231.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of Syt1-C2AB-SNAREpin complexes on the surface of negatively charged lipid nanotube flipped along z-axis. 10.4A resolution.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.49 Å/pix.
x 420 pix.
= 625.8 Å		1.49 Å/pix.
x 420 pix.
= 625.8 Å		1.49 Å/pix.
x 420 pix.
= 625.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.49 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0134 / Movie #1: 0.0134
Minimum - Maximum-0.025424857 - 0.0494302
Average (Standard dev.)0.0031392234 (±0.008239124)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 625.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.491.491.49
M x/y/z420420420
origin x/y/z0.0000.0000.000
length x/y/z625.800625.800625.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS420420420
D min/max/mean-0.0250.0490.003

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Supplemental data

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Mask #1

Fileemd_9231_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 1 used for FSC calculation

Fileemd_9231_half_map_1.map
AnnotationHalf-map 1 used for FSC calculation
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 2 used for FSC calculation

Fileemd_9231_half_map_2.map
AnnotationHalf-map 2 used for FSC calculation
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : Synaptotagmin 1 C2AB in a complex with the SNAREpin assembly boun...

EntireName: Synaptotagmin 1 C2AB in a complex with the SNAREpin assembly bound to the negatively charged phospholipid nanotube in presence of Mg2+.
Components
  • Complex: Synaptotagmin 1 C2AB in a complex with the SNAREpin assembly bound to the negatively charged phospholipid nanotube in presence of Mg2+.
    • Protein or peptide: Synaptotagmin 1
    • Protein or peptide: Vesicle-associated membrane protein 2
    • Protein or peptide: Syntaxin-1A
    • Protein or peptide: Synaptosomal-associated protein 25
    • Protein or peptide: Synaptosomal-associated protein 25

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Supramolecule #1: Synaptotagmin 1 C2AB in a complex with the SNAREpin assembly boun...

SupramoleculeName: Synaptotagmin 1 C2AB in a complex with the SNAREpin assembly bound to the negatively charged phospholipid nanotube in presence of Mg2+.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Rattus norvegicus (Norway rat)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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Macromolecule #1: Synaptotagmin 1

MacromoleculeName: Synaptotagmin 1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
SequenceString: KLGKLQYSLD YDFQNNQLLV GIIQAAELPA LDMGGTSDPY VKVFLLPDKK KKFETKVHRK TLNPVFNEQF TFKVPYSELG GKTLVMAVYD FDRFSKHDII GEFKVPMNTV DFGHVTEEWR DLQSAEKEEQ EKLGDICFSL RYVPTAGKLT VVILEAKNLK KMDVGGLSDP ...String:
KLGKLQYSLD YDFQNNQLLV GIIQAAELPA LDMGGTSDPY VKVFLLPDKK KKFETKVHRK TLNPVFNEQF TFKVPYSELG GKTLVMAVYD FDRFSKHDII GEFKVPMNTV DFGHVTEEWR DLQSAEKEEQ EKLGDICFSL RYVPTAGKLT VVILEAKNLK KMDVGGLSDP YVKIHLMQNG KRLKKKKTTI KKNTLNPYYN ESFSFEVPFE QIQKVQVVVT VLDYDKIGKN DAIGKVFVGY NSTGAELRHW SDMLANPRRP IAQWHTLQVE EEVDAMLAVK K

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Macromolecule #2: Vesicle-associated membrane protein 2

MacromoleculeName: Vesicle-associated membrane protein 2 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
SequenceString:
GSNRRLQQTQ AQVDEVVDIM RVNVDKVLER DQKLSELDDR ADALQAGASQ FETSAAKLKR KYW

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Macromolecule #3: Syntaxin-1A

MacromoleculeName: Syntaxin-1A / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
SequenceString:
MALSEIETRH SEIIKLENSI RELHDMFMDM AMLVESQGEM IDRIEYNVEH AVDYVERAVS DTKKAVK

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Macromolecule #4: Synaptosomal-associated protein 25

MacromoleculeName: Synaptosomal-associated protein 25 / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
SequenceString:
MRNELEEMQR RADQLADESL ESTRRMLQLV EESKDAGIRT LVMLDEQGEQ LDRVEEGMNH INQDMKEAEK NLKDLGK

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Macromolecule #5: Synaptosomal-associated protein 25

MacromoleculeName: Synaptosomal-associated protein 25 / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO
SequenceString:
MARENEMDEN LEQVSGIIGN LRHMALDMGN EIDTQNRQID RIMEKADSNK TRIDEANQRA TKMLG

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI TECNAI F20
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average exposure time: 9.9 sec. / Average electron dose: 44.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 7.3 Å
Applied symmetry - Helical parameters - Δ&Phi: 78.48 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 10.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 2082
Startup modelType of model: INSILICO MODEL / In silico model: Featureless hollow cylinder
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

PDB-6mti:
Synaptotagmin-1 C2A, C2B domains and SNARE-pin proteins (5CCI) individually docked into Cryo-EM map of C2AB-SNARE complexes helically organized on lipid nanotube surface in presence of Mg2+

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