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- EMDB-25740: Composite map of Upright Munc13-1 C1-C2B-MUN-C2C molecule spannin... -

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Basic information

Entry
Database: EMDB / ID: EMD-25740
TitleComposite map of Upright Munc13-1 C1-C2B-MUN-C2C molecule spanning two lipid bilayers
Map dataComposite map created in UCSF Chimera by combining two maps from independent, focused 3D classifications using the "vop maximum" command
Sample
  • Complex: 2D crystal of Munc13-1 C1-C2B-MUN-C2C domains between two lipid bilayers.
    • Protein or peptide: Protein unc-13 homolog A
KeywordsSynaptic Transmission / Munc13 / Membrane Fusion / EXOCYTOSIS
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)
Methodsubtomogram averaging / cryo EM / Resolution: 10.0 Å
AuthorsGrushin K / Sindelar CV
Funding support United States, 2 items
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, 2021-
Header (metadata) releaseFeb 9, 2022-
Map releaseFeb 9, 2022-
UpdateFeb 28, 2024-
Current statusFeb 28, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.154
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.154
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-7t7x
  • Surface level: 0.154
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_25740.png

Downloads & links

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Map

FileDownload / File: emd_25740.map.gz / Format: CCP4 / Size: 33 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite map created in UCSF Chimera by combining two maps from independent, focused 3D classifications using the "vop maximum" command
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.1 Å/pix.
x 206 pix.
= 432.6 Å		2.1 Å/pix.
x 207 pix.
= 434.7 Å		2.1 Å/pix.
x 203 pix.
= 426.3 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 2.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.154 / Movie #1: 0.154
Minimum - Maximum-0.091152094 - 1.0482712
Average (Standard dev.)0.0017767854 (±0.024750756)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-8-7-8
Dimensions207203206
Spacing203207206
CellA: 426.3 Å / B: 434.69998 Å / C: 432.59998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.12.12.1
M x/y/z203207206
origin x/y/z0.0000.0000.000
length x/y/z426.300434.700432.600
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ180180180
MAP C/R/S123
start NC/NR/NS-7-8-8
NC/NR/NS203207206
D min/max/mean-0.0911.0480.002

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Supplemental data

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Sample components

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Entire : 2D crystal of Munc13-1 C1-C2B-MUN-C2C domains between two lipid b...

EntireName: 2D crystal of Munc13-1 C1-C2B-MUN-C2C domains between two lipid bilayers.
Components
  • Complex: 2D crystal of Munc13-1 C1-C2B-MUN-C2C domains between two lipid bilayers.
    • Protein or peptide: Protein unc-13 homolog A

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Supramolecule #1: 2D crystal of Munc13-1 C1-C2B-MUN-C2C domains between two lipid b...

SupramoleculeName: 2D crystal of Munc13-1 C1-C2B-MUN-C2C domains between two lipid bilayers.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 130 KDa

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Macromolecule #1: Protein unc-13 homolog A

MacromoleculeName: Protein unc-13 homolog A / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 130.895867 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GPLGSEFMAG ITSALASSTL NNEELKNHVY KKTLQALIYP ISCTTPHNFE VWTATTPTYC YECEGLLWGI ARQGMRCTEC GVKCHEKCQ DLLNADCLQR AAEKSSKHGA EDRTQNIIMV LKDRMKIRER NKPEIFELIQ EVFAVTKSAH TQQMKAVKQS V LDGTSKWS ...String:
GPLGSEFMAG ITSALASSTL NNEELKNHVY KKTLQALIYP ISCTTPHNFE VWTATTPTYC YECEGLLWGI ARQGMRCTEC GVKCHEKCQ DLLNADCLQR AAEKSSKHGA EDRTQNIIMV LKDRMKIRER NKPEIFELIQ EVFAVTKSAH TQQMKAVKQS V LDGTSKWS AKISITVVCA QGLQAKDKTG SSDPYVTVQV GKTKKRTKTI YGNLNPVWEE NFHFECHNSS DRIKVRVWDE DD DIKSRVK QRFKRESDDF LGQTIIEVRT LSGEMDVWYN LDKRTDKSAV SGAIRLHISV EIKGEEKVAP YHVQYTCLHE NLF HFVTDV QNNGVVKIPD AKGDDAWKVY YDETAQEIVD EFAMRYGVES IYQAMTHFAC LSSKYMCPGV PAVMSTLLAN INAY YAHTT ASTNVSASDR FAASNFGKER FVKLLDQLHN SLRIDLSMYR NNFPASSPER LQDLKSTVDL LTSITFFRMK VQELQ SPPR ASQVVKDCVK ACLNSTYEYI FNNCHELYGR EYQTDPAKKG EVPPEEQGPS IKNLDFWSKL ITLIVSIIEE DKNSYT PCL NQFPQELNVG KISAEVMWSL FAQDMKYAME EHDKHRLCKS ADYMNLHFKV KWLYNEYVAE LPTFKDRVPE YPAWFEP FV IQWLDENEEV SRDFLHGALE RDKKDGFQQT SEHALFSCSV VDVFSQLNQS FEIIKKLECP DPQIVGHYMR RFAKTISN V LLQYADIVSK DFASYCSKEK EKVPCILMNN TQQLRVQLEK MFEAMGGKEL DAEASGTLKE LQVKLNNVLD ELSHVFATS FQPHIEECVR QMGDILSQVK GTGNVPASAC SSVAQDADNV LQPIMDLLDS NLTLFAKICE KTVLKRVLKE LWKLVMNTME RTIVLPPEF LSKLKDHMVR EEAKSLTPKQ CAVVELALDT IKQYFHAGGV GLKKTFLEKS PDLQSLRYAL SLYTQATDLL I KTFVQTQS AQGSGVEDPV GEVSVHVELF THPGTGEQKV TVKVVAANDL KWQTSGIFRP FIEVNIVGPQ LSDKKRKFAT KS KNNSWAP KYNESFQFSL SADAGPECYE LQVCVKDYCF AREDRTVGLA VLQLRELAQR GSAACWLPLG RRIHMDDTGL TVL RILSQR SNDEVAKEFV KLKSDTRSAE EGGAAPAP

UniProtKB: Protein unc-13 homolog A, Protein unc-13 homolog A

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation state2D array

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Sample preparation

BufferpH: 7.4
Component:
ConcentrationName
20.0 mMMOPS
150.0 mMpotassium chloride
1.0 mMEDTA
0.5 mMTCEP
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 5 sec before plunging, blot force -1.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 3.1 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 5.0 µm / Nominal defocus min: 3.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 10.0 Å / Resolution method: OTHER / Software - Name: RELION (ver. 3.1)
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.
Number subtomograms used: 126711
ExtractionNumber tomograms: 62 / Number images used: 70467
Details: Particles were extracted and refined using Warp/M software
Final 3D classificationNumber classes: 8
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)

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Atomic model buiding 1

DetailsModel 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.
RefinementProtocol: FLEXIBLE FIT
Output model

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

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