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- PDB-7t7c: The hexagonal organization of Munc13-1 C1-C2B-MUN-C2C domains bet... -

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Basic information

Entry
Database: PDB / ID: 7t7c
TitleThe hexagonal organization of Munc13-1 C1-C2B-MUN-C2C domains between lipid bilayers
ComponentsProtein unc-13 homolog A Chimera
KeywordsEXOCYTOSIS / Synaptic Transmission / Munc13 / Membrane Fusion
Function / homology
Function and homology information


dense core granule priming / neuronal dense core vesicle exocytosis / diacylglycerol binding / regulation of synaptic vesicle priming / presynaptic dense core vesicle exocytosis / synaptic vesicle docking / positive regulation of glutamate receptor signaling pathway / synaptic vesicle maturation / presynaptic active zone cytoplasmic component / positive regulation of synaptic plasticity ...dense core granule priming / neuronal dense core vesicle exocytosis / diacylglycerol binding / regulation of synaptic vesicle priming / presynaptic dense core vesicle exocytosis / synaptic vesicle docking / positive regulation of glutamate receptor signaling pathway / synaptic vesicle maturation / presynaptic active zone cytoplasmic component / positive regulation of synaptic plasticity / innervation / neurotransmitter secretion / regulation of short-term neuronal synaptic plasticity / regulation of amyloid precursor protein catabolic process / positive regulation of neurotransmitter secretion / syntaxin-1 binding / syntaxin binding / synaptic vesicle priming / Golgi-associated vesicle / neuromuscular junction development / spectrin binding / presynaptic active zone / synaptic vesicle exocytosis / calyx of Held / excitatory synapse / amyloid-beta metabolic process / SNARE binding / synaptic membrane / synaptic transmission, glutamatergic / long-term synaptic potentiation / neuromuscular junction / terminal bouton / phospholipid binding / synaptic vesicle membrane / presynapse / presynaptic membrane / cell differentiation / calmodulin binding / neuron projection / protein domain specific binding / axon / glutamatergic synapse / synapse / calcium ion binding / protein-containing complex binding / protein-containing complex / identical protein binding / plasma membrane
Similarity search - Function
Mammalian uncoordinated homology 13, domain 2 / Protein Unc-13 / Protein Unc-13, C2B domain / Munc13-homology domain 2 (MHD2) profile. / MUN domain / Munc13 homology 1 / MUN domain / Munc13-homology domain 1 (MHD1) profile. / Domain of Unknown Function (DUF1041) / Phorbol esters/diacylglycerol binding domain (C1 domain) ...Mammalian uncoordinated homology 13, domain 2 / Protein Unc-13 / Protein Unc-13, C2B domain / Munc13-homology domain 2 (MHD2) profile. / MUN domain / Munc13 homology 1 / MUN domain / Munc13-homology domain 1 (MHD1) profile. / Domain of Unknown Function (DUF1041) / Phorbol esters/diacylglycerol binding domain (C1 domain) / Zinc finger phorbol-ester/DAG-type signature. / C2 domain / Protein kinase C conserved region 2 (CalB) / Zinc finger phorbol-ester/DAG-type profile. / Protein kinase C conserved region 1 (C1) domains (Cysteine-rich domains) / Protein kinase C-like, phorbol ester/diacylglycerol-binding domain / C2 domain / C2 domain profile. / C1-like domain superfamily / C2 domain superfamily
Similarity search - Domain/homology
(mimic poison frog) hypothetical protein / Protein unc-13 homolog A
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 10 Å
AuthorsGrushin, K. / Sindelar, C.V.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)NIH DK 027044 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)NIH GM 110530 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: Munc13 structural transitions and oligomers that may choreograph successive stages in vesicle priming for neurotransmitter release.
Authors: Kirill Grushin / R Venkat Kalyana Sundaram / Charles V Sindelar / James E Rothman /
Abstract: How can exactly six SNARE complexes be assembled under each synaptic vesicle? Here we report cryo-EM crystal structures of the core domain of Munc13, the key chaperone that initiates SNAREpin ...How can exactly six SNARE complexes be assembled under each synaptic vesicle? Here we report cryo-EM crystal structures of the core domain of Munc13, the key chaperone that initiates SNAREpin assembly. The functional core of Munc13, consisting of C1-C2B-MUN-C2C (Munc13C) spontaneously crystallizes between phosphatidylserine-rich bilayers in two distinct conformations, each in a radically different oligomeric state. In the open conformation (state 1), Munc13C forms upright trimers that link the two bilayers, separating them by ∼21 nm. In the closed conformation, six copies of Munc13C interact to form a lateral hexamer elevated ∼14 nm above the bilayer. Open and closed conformations differ only by a rigid body rotation around a flexible hinge, which when performed cooperatively assembles Munc13 into a lateral hexamer (state 2) in which the key SNARE assembly-activating site of Munc13 is autoinhibited by its neighbor. We propose that each Munc13 in the lateral hexamer ultimately assembles a single SNAREpin, explaining how only and exactly six SNARE complexes are templated. We suggest that state 1 and state 2 may represent two successive states in the synaptic vesicle supply chain leading to "primed" ready-release vesicles in which SNAREpins are clamped and ready to release (state 3).
History
DepositionDec 15, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 9, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 23, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.pdbx_database_id_PubMed ..._citation.journal_volume / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Feb 28, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Assembly

Deposited unit
A: Protein unc-13 homolog A Chimera
B: Protein unc-13 homolog A Chimera
C: Protein unc-13 homolog A Chimera
D: Protein unc-13 homolog A Chimera
E: Protein unc-13 homolog A Chimera
F: Protein unc-13 homolog A Chimera
G: Protein unc-13 homolog A Chimera
H: Protein unc-13 homolog A Chimera
I: Protein unc-13 homolog A Chimera
J: Protein unc-13 homolog A Chimera
K: Protein unc-13 homolog A Chimera
L: Protein unc-13 homolog A Chimera


Theoretical massNumber of molelcules
Total (without water)1,570,75012
Polymers1,570,75012
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, all chains
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Protein unc-13 homolog A Chimera / Munc13-1


Mass: 130895.867 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: RIMITATOR_LOCUS6943369, Unc13a / Variant: NM_022861 / Cell line (production host): ExpiHEK-293 / Production host: Homo sapiens (human) / References: UniProt: A0A822AJ50, UniProt: Q4KUS2

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: subtomogram averaging

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Sample preparation

ComponentName: 2D crystal of Munc13-1 C1-C2B-MUN-C2C domains between two lipid bilayers.
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.13 MDa / Experimental value: NO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Source (recombinant)Organism: Homo sapiens (human) / Cell: ExpiHEK-293
Buffer solutionpH: 7.4
Buffer component
IDConc.NameBuffer-ID
120 mMMOPS1
2150 mMpotassium chloride1
31 mMEDTA1
40.5 mMTCEP1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K / Details: blot for 5 sec before plunging, blot force -1

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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 defocus max: 5000 nm / Nominal defocus min: 3500 nm
Image recordingElectron dose: 3.1 e/Å2 / Avg electron dose per subtomogram: 110 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
7ISOLDE1.2.0model fitting
8UCSF ChimeraX1.2model fitting
9UCSF Chimera1.16model fitting
12RELION3.1final Euler assignment
14RELION3.13D reconstruction
CTF correctionDetails: CTF correction was performed during 3D reconstruction in RELION 3.1
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionResolution: 10 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 12149 / Symmetry type: POINT
EM volume selectionDetails: Particles were extracted and refined using Warp/M software
Num. of tomograms: 62 / Num. of volumes extracted: 36837
Atomic model buildingProtocol: FLEXIBLE FIT
Details: Model for fitting was generated by AlphaFold using the construct's amino acid sequence. Flexible fitting into corresponding densities was performed using ISOLDE tool in ChimeraX. The ...Details: Model for fitting was generated by AlphaFold using the construct's amino acid sequence. Flexible fitting into corresponding densities was performed using ISOLDE tool in ChimeraX. The resulting structures were copied and fitted as rigid bodies into the 3D map by the "fit in map" function in Chimera.

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