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- EMDB-2071: Gating movement in acetylcholine receptor analysed by time-resolv... -

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Entry
Database: EMDB / ID: 2071
TitleGating movement in acetylcholine receptor analysed by time-resolved electron cryo-microscopy
Keywordsacetylcholine receptor / freeze-trapping / asymmetric gating / allosteric mechanism
Samplenicotinic acetylcholine receptor in native postsynaptic membrane from Torpedo marmorata
SourceTorpedo marmorata / fish / marbled electric ray / image: Torpedo californica
Map dataDensity map of acetylcholine receptor
Methodhelical reconstruction, at 6.2 Å resolution
AuthorsUnwin N / Fujiyoshi Y
CitationJ. Mol. Biol., 2012, 422, 617-634

J. Mol. Biol., 2012, 422, 617-634 StrPapers
Gating movement of acetylcholine receptor caught by plunge-freezing.
Nigel Unwin / Yoshinori Fujiyoshi

DateDeposition: Apr 12, 2012 / Header (metadata) release: Apr 17, 2012 / Map release: Aug 1, 2012 / Last update: Apr 12, 2012

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.2
  • Imaged by UCSF CHIMERA
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  • Surface view colored by cylindrical radius
  • Surface level: 1.2
  • Imaged by UCSF CHIMERA
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  • Surface view with fitted model
  • Atomic models: : PDB-4aq5
  • Surface level: 1.2
  • Imaged by UCSF CHIMERA
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  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-4aq5
  • Imaged by Jmol
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Map

Fileemd_2071.map.gz (map file in CCP4 format, 10753 KB)
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AxesZ (Sec.)X (Row.)Y (Col.)
168 pix
1 Å/pix.
= 168. Å
128 pix
1 Å/pix.
= 128. Å
128 pix
1 Å/pix.
= 128. Å

Surface

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Images are generated by Spider package.

Voxel sizeX=Y=Z: 1 Å
Density
Contour Level:1.2 (by author), 1.2 (movie #1):
Minimum - Maximum-3.90023351 - 4.90560436
Average (Standard dev.)0E-8 (1)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderYXZ
Dimensions128128168
Origin000
Limit127127167
Spacing128128168
CellA: 128 Å / B: 128 Å / C: 168 Å
Alpha=beta=gamma: 90 deg.

CCP4 map header:

modeImage stored as Reals
A/pix X/Y/Z111
M x/y/z128128168
origin x/y/z0.0000.0000.000
length x/y/z128.000128.000168.000
alpha/beta/gamma90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ128128168
MAP C/R/S213
start NC/NR/NS000
NC/NR/NS128128168
D min/max/mean-3.9004.906-0.000

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Supplemental data

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Sample components

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Entire nicotinic acetylcholine receptor in native postsynaptic membrane ...

EntireName: nicotinic acetylcholine receptor in native postsynaptic membrane from Torpedo marmorata
Number of components: 1 / Oligomeric State: 5 subunits
MassTheoretical: 300 kDa / Experimental: 300 kDa
Measured by: molecular weight based on amino acid sequence data and attached sugars

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Component #1: protein, nicotinic acetylcholine receptor

ProteinName: nicotinic acetylcholine receptor / a.k.a: nicotinic receptor / Oligomeric Details: pentamer
Details: Protein is embedded in postsynaptic membrane isolated from Torpedo marmorata electric organ
Recombinant expression: No
MassTheoretical: 300 kDa / Experimental: 300 kDa
SourceSpecies: Torpedo marmorata / fish / marbled electric ray / image: Torpedo californica
Source (natural)Organelle: plasma membrane / Location in cell: plasma membrane / Cell: electrocyte cells / Organ or tissue: electric organ

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Experimental details

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Sample preparation

Specimen statehelical array
Helical parametersHand: RIGHT HANDED
Crystal grow detailsTubular membrane crystals of acetylcholine receptors grow spontaneously from isolated postsynaptic membranes when incubated in low salt buffer at 17 degrees C for two weeks
Sample solutionBuffer solution: 100 mM sodium cacodylate, 1 mM calcium chloride
pH: 7
Support film300 mesh copper grid with pre-irradiated thick holey carbon support, glow discharged in amylamine atmosphere
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 120 K / Humidity: 85 %
Method: Blot until applied droplet loses contact with filter paper (indicated by loss of transparency; typically 6s)
Time resolved state: Vitrified within 10ms of exposure to acetylcholine (applied as the grid is being plunged,using a fine, focussed spray positioned about 1cm above the ethane surface)
Details: Vitrification carried out at an ambient temperature of 8 degrees C

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Electron microscopy imaging

ImagingMicroscope: JEOL 3000SFF / Date: Nov 1, 2005
Details: Standard low dose imaging of specimens over holes in the carbon support film
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 25 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 40000 X (nominal), 38500 X (calibrated)
Astigmatism: Objective lens astigmatism was corrected based on appearance of carbon film at 250,000 times magnification
Cs: 1.6 mm / Imaging mode: BRIGHT FIELD / Defocus: 900 - 2000 nm
Specimen HolderHolder: Top-entry holder for liquid helium cooled stage (the temperature of the specimen in this holder is usually at 4K)
Model: OTHER / Temperature: 10 K ( 10 - 20 K)
CameraDetector: KODAK SO-163 FILM

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Image acquisition

Image acquisitionNumber of digital images: 111 / Scanner: OTHER / Sampling size: 2.5 microns / Bit depth: 16 / OD range: 1
Details: All images recorded on film, developed in Kodak d19 developer

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Image processing

ProcessingMethod: helical reconstruction
Details: Alignment and distortion correction of each tube image was done using a segmental Fourier-Bessel method (Beroukhim & Unwin (1997) Ultramicroscopy, 70:57-81) with 50% overlap between successive segments
3D reconstructionResolution method: FSC 0.5
Details: Final maps were calculated from 111 tube images(closed class) and 123 tube images (open class)
Software: MRC, and, own, programs / Algorithm: Standard Fourier-Bessel synthesis / CTF correction: Each tube image / Resolution: 6.2 Å

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Atomic model buiding

Modeling #1Software: DireX / Refinement protocol: flexible / Refinement space: REAL
Details: Protocol: Maximisation of correlation between experimental densities and atomic model, using a deformable elastic network algorithm. Identical refinement procedures were applied to both density maps. The fits were validated by applying the same refinement procedures to independent density maps calculated from half-datasets
Input PDB model: 2BG9
Chain ID: A, B, C, D, E
Output model

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