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- EMDB-2039: negative stain Electron Microscopy of the N-Ethylmaleimide Sensit... -

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Basic information

Entry
Database: EMDB / ID: EMD-2039
Titlenegative stain Electron Microscopy of the N-Ethylmaleimide Sensitive Factor (NSF)
Map dataNSF-AMP-PNP
Sample
  • Sample: NSF-AMP-PNP
  • Protein or peptide: N-Ethylmaleimide Sensitive Factor
KeywordsNSF / N-Ethylmaleimide Sensitive Factor
Biological speciesCricetulus griseus (Chinese hamster)
Methodsingle particle reconstruction / Resolution: 20.0 Å
AuthorsMoeller A / Zhao C / Fried MG / Wilson-Kubalek EM / Carragher B / Whiteheart SW
CitationJournal: J Struct Biol / Year: 2012
Title: Nucleotide-dependent conformational changes in the N-Ethylmaleimide Sensitive Factor (NSF) and their potential role in SNARE complex disassembly.
Authors: Arne Moeller / Chunxia Zhao / Michael G Fried / Elizabeth M Wilson-Kubalek / Bridget Carragher / Sidney W Whiteheart /
Abstract: Homohexameric, N-Ethylmaleimide Sensitive Factor (NSF) disassembles Soluble NSF Attachment Protein Receptor (SNARE) complexes after membrane fusion, an essential step in vesicular trafficking. NSF ...Homohexameric, N-Ethylmaleimide Sensitive Factor (NSF) disassembles Soluble NSF Attachment Protein Receptor (SNARE) complexes after membrane fusion, an essential step in vesicular trafficking. NSF contains three domains (NSF-N, NSF-D1, and NSF-D2), each contributing to activity. We combined electron microscopic (EM) analysis, analytical ultracentrifugation (AU) and functional mutagenesis to visualize NSF's ATPase cycle. 3D density maps show that NSF-D2 remains stable, whereas NSF-N undergoes large conformational changes. NSF-Ns splay out perpendicular to the ADP-bound hexamer and twist upwards upon ATP binding, producing a more compact structure. These conformations were confirmed by hydrodynamic, AU measurements: NSF-ATP sediments faster with a lower frictional ratio (f/f(0)). Hydrodynamic analyses of NSF mutants, with specific functional defects, define the structures underlying these conformational changes. Mapping mutations onto our 3D models allows interpretation of the domain movement and suggests a mechanism for NSF binding to and disassembly of SNARE complexes.
History
DepositionJan 23, 2012-
Header (metadata) releaseFeb 15, 2012-
Map releaseFeb 15, 2012-
UpdateDec 11, 2013-
Current statusDec 11, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

2039.jpg

Downloads & links

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Map

FileDownload / File: emd_2039.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNSF-AMP-PNP
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.74 Å/pix.
x 80 pix.
= 219.2 Å		2.74 Å/pix.
x 80 pix.
= 219.2 Å		2.74 Å/pix.
x 80 pix.
= 219.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.74 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.015
Minimum - Maximum-0.02587483 - 0.09419183
Average (Standard dev.)0.00251314 (±0.00791607)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions808080
Spacing808080
CellA=B=C: 219.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.742.742.74
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z219.200219.200219.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS808080
D min/max/mean-0.0260.0940.003

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Supplemental data

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Sample components

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Entire : NSF-AMP-PNP

EntireName: NSF-AMP-PNP
Components
  • Sample: NSF-AMP-PNP
  • Protein or peptide: N-Ethylmaleimide Sensitive Factor

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Supramolecule #1000: NSF-AMP-PNP

SupramoleculeName: NSF-AMP-PNP / type: sample / ID: 1000 / Oligomeric state: hexamer / Number unique components: 1

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Macromolecule #1: N-Ethylmaleimide Sensitive Factor

MacromoleculeName: N-Ethylmaleimide Sensitive Factor / type: protein_or_peptide / ID: 1 / Name.synonym: NSF / Recombinant expression: No
Source (natural)Organism: Cricetulus griseus (Chinese hamster) / synonym: Chinese hamster

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Experimental details

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Structure determination

Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
DateMay 20, 2011
Image recordingNumber real images: 858 / Average electron dose: 25 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: -2.0 µm / Nominal defocus min: -0.2 µm / Nominal magnification: 62000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: whole image
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Xmipp, Appion, Imagic / Number images used: 12978

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