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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 / homologyAAA+ lid domain / CDC48, N-terminal subdomain / AAA-protein family signature. / Cell division protein 48 (CDC48), domain 2 / Cell division protein 48 (CDC48), N-terminal domain / ATPase family associated with various cellular activities (AAA) / Vesicle-fusing ATPase / CDC48 domain 2-like superfamily / P-loop containing nucleoside triphosphate hydrolase / Aspartate decarboxylase-like domain superfamily ...AAA+ lid domain / CDC48, N-terminal subdomain / AAA-protein family signature. / Cell division protein 48 (CDC48), domain 2 / Cell division protein 48 (CDC48), N-terminal domain / ATPase family associated with various cellular activities (AAA) / Vesicle-fusing ATPase / CDC48 domain 2-like superfamily / P-loop containing nucleoside triphosphate hydrolase / Aspartate decarboxylase-like domain superfamily / CDC48, domain 2 / ATPase, AAA-type, conserved site / ATPase, AAA-type, core / AAA+ ATPase domain / SNARE complex disassembly / vesicle-fusing ATPase / positive regulation of receptor recycling / syntaxin-1 binding / ionotropic glutamate receptor binding / SNARE binding / potassium ion transport / ATPase activity, coupled / PDZ domain binding / positive regulation of protein catabolic process / intracellular protein transport / midbody / ATPase activity / protein-containing complex binding / myelin sheath / protein kinase binding / ATP binding / identical protein binding / plasma membrane / metal ion binding / cytosol / Vesicle-fusing ATPase
Function and homology information
Specimen sourceCricetulus griseus (Chinese hamster)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 4.2 Å resolution
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
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Dec 5, 2014 / Release: Jan 28, 2015
RevisionDateData content typeGroupCategoryItemProviderType
1.0Jan 28, 2015Structure modelrepositoryInitial release
1.1Feb 11, 2015Structure modelDatabase references
1.2Jul 18, 2018Structure modelData collectionem_image_scans / em_software_em_software.image_processing_id / _em_software.name

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Structure visualization

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  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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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
Polyers497,4456
Non-polymers5,57911
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein/peptide
Vesicle-fusing ATPase / / 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: (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 / Formula: C10H16N5O13P3 / Adenosine triphosphate / Comment: ATP (energy-carrying molecule) *YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 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
Details: 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
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
MicroscopyMicroscope model: FEI POLARA 300 / Date: Feb 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 / 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)
RadiationDiffraction protocol: SINGLE WAVELENGTH / Monochromatic or laue m 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
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 50781 / Nominal pixel size: 1.2156 / Actual pixel size: 1.2156
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 buildingDetails: REFINEMENT PROTOCOL--flexible DETAILS--D2 domain was from crystal structure 1NSF. D1 domain was built de novo.
Ref protocol: FLEXIBLE FIT / Ref space: RECIPROCAL / Target criteria: R-factor
Atomic model buildingPDB-ID: 1NSF
Pdb chain ID: A
RefineOverall SU ML: 0.78 / Sigma F: 0.12 / Overall phase error: 37.83 / Stereochemistry target values: ML
Solvent computationSolvent shrinkage radii: 0.9 Å / Solvent vdw probe radii: 1.11 Å / Solvent model details: FLAT BULK SOLVENT MODEL
Displacement parametersB iso max: 375.18 Å2 / B iso mean: 200.7475 Å2 / B iso min: 120.8 Å2
Least-squares processR factor R free: 0.3023 / R factor R work: 0.2808 / R factor obs: 0.2819 / Highest resolution: 4.2 Å / Lowest resolution: 311.194 Å / Number reflection R free: 42485 / Number reflection R work: 808728 / Number reflection obs: 851213 / Percent reflection R free: 4.99 / Percent reflection obs: 99.91
Refine hist #LASTHighest resolution: 4.2 Å / Lowest resolution: 311.194 Å
Number of atoms included #LASTProtein: 21168 / Nucleic acid: 0 / Ligand: 341 / Solvent: 0 / Total: 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
Refine LS shell

Refine ID: ELECTRON MICROSCOPY / Total number of bins used: 30

Highest resolutionR factor R freeR factor R workLowest resolutionNumber reflection R freeNumber reflection R workNumber reflection allPercent reflection obs
4.20030.50930.47864.248114042696128365100.0
4.24810.46390.44254.298114642711528579100.0
4.29810.46270.42634.350513922727328665100.0
4.35050.41500.40484.405614042676128165100.0
4.40560.39980.38964.463514282697928407100.0
4.46350.39750.38004.524714282670128129100.0
4.52470.39180.37454.589313802741728797100.0
4.58930.38710.36644.657814522676728219100.0
4.65780.38520.35724.730614322718928621100.0
4.73060.36080.35144.808214162680528221100.0
4.80820.38120.34774.891114462707928525100.0
4.89110.35780.34304.980113922702728419100.0
4.98010.35500.33975.075814042673728141100.0
5.07580.36380.33625.179513552725328608100.0
5.17950.36170.33385.292114512708428535100.0
5.29210.36360.34445.415214162634927765100.0
5.41520.38060.34335.550614402740928849100.0
5.55060.38960.34615.700713862697428360100.0
5.70070.36780.35225.868514762696528441100.0
5.86850.37450.35446.057914042677628180100.0
6.05790.36500.35306.274513922700428396100.0
6.27450.36020.33926.525713802684228222100.0
6.52570.34800.32436.822714282736428792100.0
6.82270.33270.29957.182514642671028174100.0
7.18250.33760.30677.632513622699928361100.0
7.63250.28760.26328.221814042707728481100.0
8.22180.24760.23069.049314402681328253100.0
9.04930.19990.177810.358814272694228369100.0
10.35880.17690.165413.05111426267812820799.0
13.05110.20470.1794312.05141392265752796799.0

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