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- EMDB-6205: Structure of ADP-bound N-ethylmaleimide sensitive factor determin... -

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Basic information

Entry
Database: EMDB / ID: EMD-6205
TitleStructure of ADP-bound N-ethylmaleimide sensitive factor determined by single particle cryoelectron microscopy
Map dataMap of ADP-bound N-ethylmaleimide sensitive factor. This map is unsharpened and unfiltered. The map was normalized using the program MAPMAN.
Sample
  • Sample: ADP-bound N-ethylmaleimide sensitive factor
  • Protein or peptide: N-ethylmaleimide sensitive factor
KeywordsATPases associated with diverse cellular activities
Function / homology
Function and homology information


SNARE complex disassembly / ATP-dependent protein disaggregase activity / 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 ...SNARE complex disassembly / ATP-dependent protein disaggregase activity / 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 / Golgi apparatus / ATP hydrolysis activity / ATP binding / identical protein binding / metal ion binding / plasma membrane / cytosol
Similarity search - Function
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 ...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
Vesicle-fusing ATPase
Similarity search - Component
Biological speciesCricetulus griseus (Chinese hamster)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.6 Å
AuthorsZhao M / Wu S / Zhou Q / Vivona S / Cipriano DJ / Cheng Y / Brunger AT
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, 2014-
Header (metadata) releaseJan 28, 2015-
Map releaseJan 28, 2015-
UpdateFeb 11, 2015-
Current statusFeb 11, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 6
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 6
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j95
  • Surface level: 6
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6205.png

Downloads & links

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Map

FileDownload / File: emd_6205.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of ADP-bound N-ethylmaleimide sensitive factor. This map is unsharpened and unfiltered. The map was normalized using the program MAPMAN.
Voxel sizeX=Y=Z: 2.4312 Å
Density
Contour LevelBy AUTHOR: 6.0 / Movie #1: 6
Minimum - Maximum-5.15959406 - 13.842989920000001
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 311.1936 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.4312031252.4312031252.431203125
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z311.194311.194311.194
α/β/γ90.00090.00090.000
start NX/NY/NZ-72-72-72
NX/NY/NZ145145145
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-5.16013.843-0.000

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Supplemental data

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Supplemental map: EMD-6205 ADP NSF sharpened -479.map

FileEMD-6205_ADP_NSF_sharpened_-479.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : ADP-bound N-ethylmaleimide sensitive factor

EntireName: ADP-bound N-ethylmaleimide sensitive factor
Components
  • Sample: ADP-bound N-ethylmaleimide sensitive factor
  • Protein or peptide: N-ethylmaleimide sensitive factor

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Supramolecule #1000: ADP-bound N-ethylmaleimide sensitive factor

SupramoleculeName: ADP-bound N-ethylmaleimide sensitive factor / type: sample / ID: 1000 / Oligomeric state: hexamer / Number unique components: 1
Molecular weightTheoretical: 500 KDa

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Macromolecule #1: N-ethylmaleimide sensitive factor

MacromoleculeName: N-ethylmaleimide sensitive factor / type: protein_or_peptide / ID: 1 / Name.synonym: NSF / Number of copies: 6 / Oligomeric state: hexamer / Recombinant expression: Yes
Source (natural)Organism: Cricetulus griseus (Chinese hamster) / synonym: Chinese hamster
Molecular weightTheoretical: 83 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3)-RIL / Recombinant plasmid: pPROEX-1
SequenceUniProtKB: Vesicle-fusing ATPase

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration15 mg/mL
BufferpH: 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
GridDetails: Holey carbon on top of 400 mesh copper grid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 90 K / Instrument: FEI VITROBOT MARK I / Method: Blot for 3.5 seconds before plunging.

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.3 mm / Nominal defocus max: -2.8 µm / Nominal defocus min: -1.8 µm / Nominal magnification: 31000
Sample stageSpecimen holder model: OTHER
DateJan 14, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Average electron dose: 44 e/Å2
Details: Gatan K2 Summit in super-resolution counting mode. Motion correction as described in Li et al. (2013) Nature Methods.
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionResolution.type: BY AUTHOR / Resolution: 7.6 Å / Resolution method: OTHER / Software - Name: RELION / Number images used: 12830
Details3D classification, refinement, and reconstruction were performed using RELION.

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Atomic model buiding 1

Initial modelPDB ID:

1nsf


Chain - Chain ID: A
SoftwareName: Chimera, PHENIX
DetailsD2 domain of NSF was from crystal structure 1NSF. D1 domain of NSF was from related entry EMD-6204.
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT / Target criteria: R-factor
Output model

PDB-3j95:
Structure of ADP-bound N-ethylmaleimide sensitive factor determined by single particle cryoelectron microscopy

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