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- PDB-7t7x: Munc13-1 C1-C2B-MUN-C2C Upright conformation spanning two lipid b... -

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Basic information

Entry
Database: PDB / ID: 7t7x
TitleMunc13-1 C1-C2B-MUN-C2C Upright conformation spanning two lipid bilayers
ComponentsProtein unc-13 homolog A
KeywordsEXOCYTOSIS / Synaptic Transmission / Munc13 / Membrane Fusion
Function / homology
Function and homology information


dense core granule priming / neuronal dense core vesicle exocytosis / diacylglycerol binding / presynaptic dense core vesicle exocytosis / synaptic vesicle docking / regulation of synaptic vesicle priming / synaptic vesicle maturation / positive regulation of glutamate receptor signaling pathway / presynaptic active zone cytoplasmic component / positive regulation of synaptic plasticity ...dense core granule priming / neuronal dense core vesicle exocytosis / diacylglycerol binding / presynaptic dense core vesicle exocytosis / synaptic vesicle docking / regulation of synaptic vesicle priming / synaptic vesicle maturation / positive regulation of glutamate receptor signaling pathway / presynaptic active zone cytoplasmic component / positive regulation of synaptic plasticity / innervation / positive regulation of dendrite extension / neurotransmitter secretion / regulation of short-term neuronal synaptic plasticity / regulation of amyloid precursor protein catabolic process / syntaxin binding / syntaxin-1 binding / positive regulation of neurotransmitter secretion / synaptic vesicle priming / Golgi-associated vesicle / neuromuscular junction development / spectrin binding / presynaptic active zone / synaptic vesicle exocytosis / excitatory synapse / calyx of Held / amyloid-beta metabolic process / SNARE binding / synaptic transmission, glutamatergic / synaptic membrane / 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 / calcium ion binding / synapse / 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. / Protein kinase C conserved region 2 (CalB) / C2 domain / 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
Protein unc-13 homolog A / 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


Theoretical massNumber of molelcules
Total (without water)130,8961
Polymers130,8961
Non-polymers00
Water00
1


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

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Components

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


Mass: 130895.867 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Unc13a, Unc13h1, Unc13a / Variant: NM_022861 / Cell line (production host): ExpiHEK-293 / Production host: Homo sapiens (human) / References: UniProt: Q62768, 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
13RELION3.1final Euler assignment
15RELION3.13D reconstruction
CTF correctionDetails: CTF correction was performed during 3D reconstruction in RELION 3.1
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 10 Å / Resolution method: OTHER / Num. of particles: 126711
Details: Combined map of best classes from two independent 3D classifications in RELION 3.1 of selected regions without angular searches using C3 symmetry expanded dataset (126711 subtomograms) and ...Details: Combined map of best classes from two independent 3D classifications in RELION 3.1 of selected regions without angular searches using C3 symmetry expanded dataset (126711 subtomograms) and separation into 8 classes.
Symmetry type: POINT
EM volume selectionDetails: Particles were extracted and refined using Warp/M software
Num. of tomograms: 62 / Num. of volumes extracted: 70467
Atomic model buildingProtocol: FLEXIBLE FIT
Details: Model for fitting was generated by AlphaFold using the construct's amino acid sequence. Flexible fitting into 3D map densities was performed using ISOLDE tool in ChimeraX.

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