[English] 日本語
Yorodumi
- PDB-3j97: Structure of 20S supercomplex determined by single particle cryoe... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 3j97
TitleStructure of 20S supercomplex determined by single particle cryoelectron microscopy (State II)
Components
  • Alpha-soluble NSF attachment protein
  • Synaptosomal-associated protein 25SNAP25
  • Syntaxin-1A
  • Vesicle-associated membrane protein 2Vesicle-associated membrane protein
  • Vesicle-fusing ATPase
KeywordsHYDROLASE / vesicle trafficking
Function / homology
Function and homology information


soluble NSF attachment protein activity / SNARE complex disassembly / regulation of delayed rectifier potassium channel activity / anchored component of presynaptic membrane / myosin head/neck binding / eosinophil degranulation / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin II complex / exocytic insertion of neurotransmitter receptor to postsynaptic membrane / synaptobrevin 2-SNAP-25-syntaxin-1a complex / growth hormone secretion ...soluble NSF attachment protein activity / SNARE complex disassembly / regulation of delayed rectifier potassium channel activity / anchored component of presynaptic membrane / myosin head/neck binding / eosinophil degranulation / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin II complex / exocytic insertion of neurotransmitter receptor to postsynaptic membrane / synaptobrevin 2-SNAP-25-syntaxin-1a complex / growth hormone secretion / positive regulation of neurotransmitter secretion / presynaptic active zone membrane / zymogen granule membrane / hormone secretion / regulation of synaptic vesicle priming / positive regulation of norepinephrine secretion / synaptobrevin 2-SNAP-25-syntaxin-1a-complexin I complex / synaptic vesicle fusion to presynaptic active zone membrane / short-term synaptic potentiation / positive regulation of catecholamine secretion / regulation of establishment of protein localization / synaptic vesicle docking / regulated exocytosis / neurotransmitter transport / protein-containing complex disassembly / storage vacuole / calcium ion-regulated exocytosis of neurotransmitter / intracellular organelle / vesicle docking / positive regulation of calcium ion-dependent exocytosis / regulation of synaptic vesicle recycling / secretion by cell / apical protein localization / protein localization to membrane / Golgi to plasma membrane protein transport / vesicle-fusing ATPase / neurotransmitter receptor internalization / regulation of vesicle-mediated transport / vesicle fusion / SNARE complex / chloride channel inhibitor activity / positive regulation of hormone secretion / SNAP receptor activity / calcium-ion regulated exocytosis / response to gravity / SNARE complex assembly / synaptic vesicle priming / integral component of synaptic vesicle membrane / positive regulation of synaptic plasticity / positive regulation of intracellular protein transport / regulation of exocytosis / membrane fusion / ATP-dependent protein binding / positive regulation of receptor recycling / actomyosin / sleep / regulation of synapse assembly / positive regulation of ATPase activity / synaptic vesicle exocytosis / modulation of excitatory postsynaptic potential / neuron projection terminus / myosin binding / syntaxin-1 binding / neurotransmitter secretion / vacuolar membrane / regulation of neuron projection development / clathrin-coated vesicle / positive regulation of exocytosis / syntaxin binding / insulin secretion / synaptic vesicle endocytosis / protein sumoylation / exocytosis / ionotropic glutamate receptor binding / voltage-gated potassium channel complex / voltage-gated potassium channel activity / synaptic transmission, glutamatergic / long-term memory / calcium channel inhibitor activity / associative learning / positive regulation of insulin secretion / somatodendritic compartment / positive regulation of excitatory postsynaptic potential / endomembrane system / long-term synaptic potentiation / axonal growth cone / SNARE binding / axonogenesis / synaptic vesicle membrane / acrosomal vesicle / photoreceptor inner segment / response to glucose / potassium ion transport / locomotory behavior / filopodium / vesicle-mediated transport / integral component of presynaptic membrane / PDZ domain binding / neuron differentiation / phospholipid binding
SNAP-25 family / Synaptobrevin/Vesicle-associated membrane protein / Target SNARE coiled-coil homology domain / NSF attachment protein / SNAP-25 domain / Synaptobrevin / CDC48, N-terminal subdomain / AAA+ ATPase domain / ATPase, AAA-type, core / CDC48, domain 2 ...SNAP-25 family / Synaptobrevin/Vesicle-associated membrane protein / Target SNARE coiled-coil homology domain / NSF attachment protein / SNAP-25 domain / Synaptobrevin / CDC48, N-terminal subdomain / AAA+ ATPase domain / ATPase, AAA-type, core / CDC48, domain 2 / Syntaxin, N-terminal domain / Syntaxin/epimorphin, conserved site / Aspartate decarboxylase-like domain superfamily / SNARE / Tetratricopeptide-like helical domain superfamily / ATPase, AAA-type, conserved site / P-loop containing nucleoside triphosphate hydrolase / v-SNARE, coiled-coil homology domain / Syntaxin 1 / Cell division protein 48 (CDC48), domain 2 / Cell division protein 48 (CDC48), N-terminal domain / ATPase family associated with various cellular activities (AAA) / AAA+ lid domain / AAA ATPase, AAA+ lid domain / Vesicle-fusing ATPase / Synaptosomal-associated protein 25 / CDC48 domain 2-like superfamily / Vesicle-associated membrane protein 2
Vesicle-associated membrane protein 2 / Synaptosomal-associated protein 25 / Alpha-soluble NSF attachment protein / Vesicle-fusing ATPase / Syntaxin-1A
Biological speciesCricetulus griseus (Chinese hamster)
Rattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.8 Å
AuthorsZhao, M. / Wu, S. / Cheng, Y. / Brunger, A.T.
CitationJournal: Nature / Year: 2015
Title: Mechanistic insights into the recycling machine of the SNARE complex.
Authors: Minglei Zhao / Shenping Wu / Qiangjun Zhou / Sandro Vivona / Daniel J Cipriano / Yifan Cheng / Axel T Brunger /
Abstract: Evolutionarily conserved SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) proteins form a complex that drives membrane fusion in eukaryotes. The ATPase NSF (N- ...Evolutionarily conserved SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptors) proteins form a complex that drives membrane fusion in eukaryotes. The ATPase NSF (N-ethylmaleimide sensitive factor), together with SNAPs (soluble NSF attachment protein), disassembles the SNARE complex into its protein components, making individual SNAREs available for subsequent rounds of fusion. Here we report structures of ATP- and ADP-bound NSF, and the NSF/SNAP/SNARE (20S) supercomplex determined by single-particle electron cryomicroscopy at near-atomic to sub-nanometre resolution without imposing symmetry. Large, potentially force-generating, conformational differences exist between ATP- and ADP-bound NSF. The 20S supercomplex exhibits broken symmetry, transitioning from six-fold symmetry of the NSF ATPase domains to pseudo four-fold symmetry of the SNARE complex. SNAPs interact with the SNARE complex with an opposite structural twist, suggesting an unwinding mechanism. The interfaces between NSF, SNAPs, and SNAREs exhibit characteristic electrostatic patterns, suggesting how one NSF/SNAP species can act on many different SNARE complexes.
Validation Report
SummaryFull reportAbout validation report
History
DepositionDec 5, 2014Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 28, 2015Provider: repository / Type: Initial release
Revision 1.1Feb 11, 2015Group: Database references
Revision 1.2Jul 18, 2018Group: Data collection / Category: em_image_scans / em_software / Item: _em_software.image_processing_id / _em_software.name

-
Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
  • Download
  • Superimposition on EM map
  • EMDB-6207
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
A: Vesicle-fusing ATPase
B: Vesicle-fusing ATPase
C: Vesicle-fusing ATPase
D: Vesicle-fusing ATPase
E: Vesicle-fusing ATPase
F: Vesicle-fusing ATPase
H: Alpha-soluble NSF attachment protein
I: Alpha-soluble NSF attachment protein
J: Alpha-soluble NSF attachment protein
G: Alpha-soluble NSF attachment protein
K: Vesicle-associated membrane protein 2
L: Syntaxin-1A
M: Synaptosomal-associated protein 25


Theoretical massNumber of molelcules
Total (without water)668,60113
Polymers668,60113
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

-
Components

#1: Protein
Vesicle-fusing ATPase / / N-ethylmaleimide-sensitive fusion protein / NEM-sensitive fusion protein / Vesicular-fusion protein ...N-ethylmaleimide-sensitive fusion protein / NEM-sensitive fusion protein / Vesicular-fusion protein NSF / N-ethylmaleimide sensitive factor


Mass: 82907.430 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Cricetulus griseus (Chinese hamster) / Gene: NSF / Production host: Escherichia coli (E. coli) / References: UniProt: P18708, vesicle-fusing ATPase
#2: Protein
Alpha-soluble NSF attachment protein / SNAP-alpha / N-ethylmaleimide-sensitive factor attachment protein alpha / alpha-SNAP


Mass: 33377.793 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Napa, Snap, Snapa / Production host: Escherichia coli (E. coli) / References: UniProt: P54921
#3: Protein Vesicle-associated membrane protein 2 / Vesicle-associated membrane protein / VAMP-2 / Synaptobrevin-2


Mass: 7231.061 Da / Num. of mol.: 1 / Fragment: UNP residues 28-89
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Vamp2, Syb2 / Production host: Escherichia coli (E. coli) / References: UniProt: P63045
#4: Protein Syntaxin-1A / Neuron-specific antigen HPC-1 / Synaptotagmin-associated 35 kDa protein / P35A


Mass: 7837.957 Da / Num. of mol.: 1 / Fragment: UNP residues 191-256
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Stx1a, Sap / Production host: Escherichia coli (E. coli) / References: UniProt: P32851
#5: Protein Synaptosomal-associated protein 25 / SNAP25 / SNAP-25 / Super protein / SUP / Synaptosomal-associated 25 kDa protein


Mass: 22576.363 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Snap25, Snap / Production host: Escherichia coli (E. coli) / References: UniProt: P60881*PLUS

-
Experimental details

-
Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

-
Sample preparation

Component
IDNameTypeDetailsParent-ID
120S supercomplex consisting of truncated neuronal SNARE complex, alpha-SNAP, and N-ethylmaleimide sensitive factor (NSF)RIBOSOMEOne hexamer of NSF + four alpha-SNAP molecules + one SNARE complex0
2N-ethylmaleimide sensitive factor1
3alpha Soluble NSF Attachment Protein1
4Syntaxin-1A1
5Synaptobrevin-21
6Synaptosomal-associated protein 25SNAP251
Molecular weightValue: 0.66 MDa / Experimental value: NO
Buffer solutionName: 50 mM Tris-Cl, 150 mM NaCl, 1 mM AMPPNP, 1 mM EDTA, 1 mM DTT, 0.05% v/v Nonident P-40
pH: 8
Details: 50 mM Tris-Cl, 150 mM NaCl, 1 mM AMPPNP, 1 mM EDTA, 1 mM DTT, 0.05% v/v Nonident P-40
SpecimenConc.: 15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Holey carbon on top of 400 mesh copper grid
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Temp: 90 K / Humidity: 100 %
Details: Blot for 3.5 seconds before plunging into liquid ethane (FEI VITROBOT MARK I).
Method: Blot for 3.5 seconds before plunging

-
Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300 / Date: Jan 28, 2014
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 31000 X / Nominal defocus max: -2800 nm / Nominal defocus min: -1800 nm / Cs: 2.3 mm / Camera length: 0 mm
Specimen holderModel: OTHER / Specimen holder type: unspecified
Image recordingElectron dose: 44 e/Å2 / Film or detector model: GATAN K2 (4k x 4k)
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

-
Processing

EM software
IDNameCategory
1PHENIXmodel fitting
2UCSF Chimeramodel fitting
3RELION3D reconstruction
CTF correctionDetails: Each particle
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 7.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 21489 / Nominal pixel size: 2.4312 Å / Actual pixel size: 2.4312 Å
Details: (Single particle details: 3D classification, refinement, and reconstruction were performed using RELION) (Single particle--Applied symmetry: C1)
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: RECIPROCAL / Target criteria: R-factor
Details: REFINEMENT PROTOCOL--flexible DETAILS--D2 domain of NSF was from crystal structure 1NSF. D1 domain of NSF was from related entry EMD-6204. N domain of NSF was from crystal structure 1QCS. ...Details: REFINEMENT PROTOCOL--flexible DETAILS--D2 domain of NSF was from crystal structure 1NSF. D1 domain of NSF was from related entry EMD-6204. N domain of NSF was from crystal structure 1QCS. aSNAP was a homology model. SNARE complex was from crystal structure 1N7S.
Atomic model building
IDPDB-IDPdb chain-ID3D fitting-ID
11NSFA1
21QCSA1
31N7SA1
41N7SB1
51N7SC1
61N7SD1
RefinementResolution: 7.804→311.194 Å / SU ML: 1.51 / σ(F): 0.19 / Phase error: 40.21 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.3109 6664 5.04 %
Rwork0.3012 125537 -
Obs0.3017 132201 99.42 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 50 Å2 / Biso mean: 50 Å2 / Biso min: 50 Å2
Refinement stepCycle: LAST / Resolution: 7.804→311.194 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms41095 0 0 0 41095
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00942100
ELECTRON MICROSCOPYf_angle_d1.56756847
ELECTRON MICROSCOPYf_chiral_restr0.0736546
ELECTRON MICROSCOPYf_plane_restr0.0067356
ELECTRON MICROSCOPYf_dihedral_angle_d15.88415533
LS refinement shell

Refinement-ID: ELECTRON MICROSCOPY / Total num. of bins used: 30

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
7.8043-7.8930.4642160.427242944510100
7.893-7.98580.48032280.417842154443100
7.9858-8.08320.48112400.407540924332100
8.0832-8.18560.48282640.412444264690100
8.1856-8.29330.42452160.41142154431100
8.2933-8.40690.44712040.402840564260100
8.4069-8.5270.43352040.404641854389100
8.527-8.65430.45782280.391442794507100
8.6543-8.78950.41032160.374341884404100
8.7895-8.93360.40832280.369442694497100
8.9336-9.08770.42842040.409141684372100
9.0877-9.2530.47882160.389641134329100
9.253-9.43090.4292760.361244544730100
9.4309-9.62350.34722160.330441124328100
9.6235-9.83270.32392160.309840714287100
9.8327-10.06150.32312150.306942734488100
10.0615-10.31310.36162380.29173990422898
10.3131-10.59190.34642250.29064289451498
10.5919-10.90360.32020.28964063426599
10.9036-11.25560.30232270.28474269449699
11.2556-11.65790.31972350.28484123435899
11.6579-12.12470.32862120.29694149436199
12.1247-12.67650.3572510.30574044429599
12.6765-13.34480.33952020.31294333453599
13.3448-14.18090.34742350.32274135437099
14.1809-15.27580.30652270.31113998422599
15.2758-16.8130.34462230.32154118434199
16.813-19.24560.31531820.33354256443899
19.2456-24.2460.3352210.31664204442599
24.246-311.44150.1451970.17764156435399

+
About Yorodumi

-
News

-
Aug 12, 2020. New: Covid-19 info

New: Covid-19 info

  • New page: Covid-19 featured information page in EM Navigator

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

-
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. New: Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242):Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force. (see PDBe EMDB page)
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is "EMD"? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB at PDBe / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary. This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated. See below links for details.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software). Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

+
Jun 16, 2017. Omokage search with filter

Omokage search with filter

  • Result of Omokage search can be filtered by keywords and the database types

Related info.:Omokage search

Read more

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more