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- PDB-3j94: Structure of ATP-bound N-ethylmaleimide sensitive factor determin... -

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Basic information

Entry
Database: PDB / ID: 3j94
TitleStructure of ATP-bound N-ethylmaleimide sensitive factor determined by single particle cryoelectron microscopy
ComponentsVesicle-fusing ATPase
KeywordsHYDROLASE / ATPases associated with diverse cellular activities
Function / homology
Function and homology information


SNARE complex disassembly / ATP-dependent protein disaggregase activity / intra-Golgi vesicle-mediated transport / Golgi to plasma membrane protein transport / Golgi stack / vesicle-fusing ATPase / syntaxin-1 binding / positive regulation of receptor recycling / ionotropic glutamate receptor binding / SNARE binding ...SNARE complex disassembly / ATP-dependent protein disaggregase activity / intra-Golgi vesicle-mediated transport / Golgi to plasma membrane protein transport / Golgi stack / vesicle-fusing ATPase / syntaxin-1 binding / positive regulation of receptor recycling / ionotropic glutamate receptor binding / SNARE binding / PDZ domain binding / intracellular protein transport / potassium ion transport / positive regulation of protein catabolic process / midbody / protein-containing complex binding / protein kinase binding / ATP hydrolysis activity / ATP binding / identical protein binding / metal ion binding / plasma membrane / cytosol
Similarity search - Function
: / NSF, AAA+ ATPase lid domain / Vesicle-fusing ATPase / Cell division protein 48 (CDC48), N-terminal domain / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48) N-terminal domain / CDC48, domain 2 / Cell division protein 48 (CDC48), domain 2 / Cell division protein 48 (CDC48) domain 2 / CDC48 domain 2-like superfamily ...: / NSF, AAA+ ATPase lid domain / Vesicle-fusing ATPase / Cell division protein 48 (CDC48), N-terminal domain / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48) N-terminal domain / CDC48, domain 2 / Cell division protein 48 (CDC48), domain 2 / Cell division protein 48 (CDC48) domain 2 / CDC48 domain 2-like superfamily / Aspartate decarboxylase-like domain superfamily / AAA ATPase, AAA+ lid domain / AAA+ lid domain / ATPase, AAA-type, conserved site / AAA-protein family signature. / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / Vesicle-fusing ATPase
Similarity search - Component
Biological speciesCricetulus griseus (Chinese hamster)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 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.
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
Revision 1.3Feb 21, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

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  • Deposited structure unit
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  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)503,02417
Polymers497,4456
Non-polymers5,57911
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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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: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: ATP-bound N-ethylmaleimide sensitive factor / Type: COMPLEX / Details: hexamer
Molecular weightValue: 0.5 MDa / Experimental value: NO
Buffer solutionName: 50 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA, 1 mM ATP, 1 mM DTT, 0.05% v/v Nonident P-40
pH: 8
Details: 50 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA, 1 mM ATP, 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

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300 / Date: Feb 28, 2014
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 31000 X / Nominal defocus max: -2800 nm / Nominal defocus min: -1800 nm / Cs: 2.3 mm / Camera length: 0 mm
Specimen holderSpecimen holder model: OTHER / Specimen holder type: unspecified
Image recordingElectron dose: 26.4 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

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Processing

EM software
IDNameCategory
1PHENIXmodel fitting
2UCSF Chimeramodel fitting
3RELION3D reconstruction
CTF correctionDetails: Each particle
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 50781 / Nominal pixel size: 1.2156 Å / Actual pixel size: 1.2156 Å
Details: Every image is the sum of 30 frames recorded using the K2 Summit. The final reconstruction was calculated from images summed from frames #2-#18. (Single particle details: 3D classification, ...Details: Every image is the sum of 30 frames recorded using the K2 Summit. The final reconstruction was calculated from images summed from frames #2-#18. (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 was from crystal structure 1NSF. D1 domain was built de novo.
Atomic model buildingPDB-ID: 1NSF

1nsf


Pdb chain-ID: A / Accession code: 1NSF / Source name: PDB / Type: experimental model
RefinementResolution: 4.2→311.194 Å / SU ML: 0.78 / σ(F): 0.12 / Phase error: 37.83 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.3023 42485 4.99 %
Rwork0.2808 808728 -
obs0.2819 851213 99.91 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 375.18 Å2 / Biso mean: 200.7475 Å2 / Biso min: 120.8 Å2
Refinement stepCycle: LAST / Resolution: 4.2→311.194 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms21168 0 341 0 21509
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00822100
ELECTRON MICROSCOPYf_angle_d1.76229952
ELECTRON MICROSCOPYf_chiral_restr0.0713551
ELECTRON MICROSCOPYf_plane_restr0.0063824
ELECTRON MICROSCOPYf_dihedral_angle_d18.4098075
LS refinement shell

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

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
4.2003-4.24810.509314040.47862696128365100
4.2481-4.29810.463914640.44252711528579100
4.2981-4.35050.462713920.42632727328665100
4.3505-4.40560.41514040.40482676128165100
4.4056-4.46350.399814280.38962697928407100
4.4635-4.52470.397514280.382670128129100
4.5247-4.58930.391813800.37452741728797100
4.5893-4.65780.387114520.36642676728219100
4.6578-4.73060.385214320.35722718928621100
4.7306-4.80820.360814160.35142680528221100
4.8082-4.89110.381214460.34772707928525100
4.8911-4.98010.357813920.3432702728419100
4.9801-5.07580.35514040.33972673728141100
5.0758-5.17950.363813550.33622725328608100
5.1795-5.29210.361714510.33382708428535100
5.2921-5.41520.363614160.34442634927765100
5.4152-5.55060.380614400.34332740928849100
5.5506-5.70070.389613860.34612697428360100
5.7007-5.86850.367814760.35222696528441100
5.8685-6.05790.374514040.35442677628180100
6.0579-6.27450.36513920.3532700428396100
6.2745-6.52570.360213800.33922684228222100
6.5257-6.82270.34814280.32432736428792100
6.8227-7.18250.332714640.29952671028174100
7.1825-7.63250.337613620.30672699928361100
7.6325-8.22180.287614040.26322707728481100
8.2218-9.04930.247614400.23062681328253100
9.0493-10.35880.199914270.17782694228369100
10.3588-13.05110.176914260.1654267812820799
13.0511-312.05140.204713920.1794265752796799

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