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Gating movement in acetylcholine receptor analysed by time-resolved electron cryo-microscopy

by helical reconstruction, at 6.2 A resolution

Movie

Orientation:

#1: Surface view with section colored by density value, Surface level: 1.2, Image by UCSF CHIMERA

#2: Surface view colored by cylindrical radius, Surface level: 1.2, Image by UCSF CHIMERA

#3: Surface view with fitted model, atomic models: PDB-4aq5, Surface level: 1.2, Image by UCSF CHIMERA

#4: Simplified surface model with fitted atomic model: PDB-4aq5, Image by Jmol

Entry
Summary
Database / IDEM DATA BANK (EMDB) / 2071
TitleGating movement in acetylcholine receptor analysed by time-resolved electron cryo-microscopy
MapDensity map of acetylcholine receptor
Samplenicotinic acetylcholine receptor in native postsynaptic membrane from Torpedo marmorata
Keywordsacetylcholine receptor, freeze-trapping, asymmetric gating, allosteric mechanism
AuthorsUnwin N, Fujiyoshi Y
DateDeposition: 2012-04-12, Header release: 2012-04-17, Map release: 2012-08-01, Last update: 2012-09-26
EMDB SitesEMDB @PDBe (EU), EMDB @RCSB (USA)
Structure Visualization
MoviesMovie Page

#1: Surface view with section colored by density value, Surface level: 1.2, Image by UCSF CHIMERA

#2: Surface view colored by cylindrical radius, Surface level: 1.2, Image by UCSF CHIMERA

#3: Surface view with fitted model, atomic models: PDB-4aq5, Surface level: 1.2, Image by UCSF CHIMERA

#4: Simplified surface model with fitted atomic model: PDB-4aq5, Image by Jmol

Supplemental images
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Related Structure Data
Related Entries

PDB-4aq5

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Fit: output model of fitting

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Article
Citation - Primary
ArticleJ. Mol. Biol., Vol. 422, Issue 5, Page 617-34, Year 2012
TitleGating movement of acetylcholine receptor caught by plunge-freezing.
AuthorsNigel Unwin, Yoshinori Fujiyoshi
MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK.
KeywordsAcetylcholine (metabolism), Allosteric Regulation, Animals, Cryoelectron Microscopy (methods), Ion Channel Gating, Models, Biological, Models, Molecular, Protein Conformation, Receptors, Cholinergic (chemistry), Torpedo
LinksPII: S0022-2836(12)00577-3, DOI: 10.1016/j.jmb.2012.07.010, PubMed: 22841691, PMC: PMC3443390
Map
Fileemd_2071.map.gz ( map file in CCP4 format, 10753 KB )
Projections & SlicesSize of images:
AxesZ (Sec.)X (Row.)Y (Col.)
168 pix
1 A/pix
= 168. A
128 pix
1 A/pix
= 128. A
128 pix
1 A/pix
= 128. A

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider package.

Density
Contour Level:1.2 (by author), 1.2 (movie #1):
Minimum - Maximum: -3.90023351 - 4.90560436
Average (Standard dev.): 0E-8 (1)
Data TypeImage stored as Reals
Space Group Number1
Map Geometry
Axis orderYXZ
Dimensions128128168
Origin000
Limit127127167
Spacing128128168
Unit CellA: 128 A, B: 128 A, C: 168 A
Alpha=beta=gamma: 90 degrees
Pixel SpacingX= Y= Z: 1 A
CCP4 map header info
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
start NC,NX/NR,NY/NS,NZ
NC,NX/NR,NY/NS,NZ
D min/max/mean-3.9004.906-0.000
Annotation DetailsDensity map of acetylcholine receptor
Supplement
Images
Images
Sample
Namenicotinic acetylcholine receptor in native postsynaptic membrane from Torpedo marmorata
Number of Components1
Oligomeric State5 subunits
Theoretical Mass0.3MDa
Mass-estimation Methodmolecular weight based on amino acid sequence data and attached sugars
Experimental Mass0.3MDa
Component #1: protein - nicotinic receptor
Scientific namenicotinic acetylcholine receptor
Common Namenicotinic receptor
Theoretical Mass0.3 MDa
Experimental Mass0.3 MDa
DetailsProtein is embedded in postsynaptic membrane isolated from Torpedo marmorata electric organ
Oligomeric Detailspentamer
Scientific Name of SpeciesTorpedo marmorata
Common Name of Speciesmarbled electric ray
NCBI taxonomy7788
Recombinant expressionNo
Natural SourceCell: electrocyte cells
Organ Or Tissue: electric organ
Organelle: plasma membrane
Cell Location: plasma membrane
Experiment
Sample Preparation
Helical ParametersHand: RIGHT HANDED
Specimen Support Details300 mesh copper grid with pre-irradiated thick holey carbon support, glow discharged in amylamine atmosphere
Specimen StatehelicalArray
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
BufferpH: 7
Details: 100 mM sodium cacodylate, 1 mM calcium chloride
Vitrification
Cryogen NameETHANE
MethodBlot until applied droplet loses contact with filter paper (indicated by loss of transparency; typically 6s)
Temperature120 Kelvin
Time Resolved StateVitrified 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)
DetailsVitrification carried out at an ambient temperature of 8 degrees C
InstrumentHOMEMADE PLUNGER
Humidity85
Imaging
MicroscopeJEOL 3000SFF
Date01-NOV-2005
DetailsStandard low dose imaging of specimens over holes in the carbon support film
Electron Gun
Electron SourceFIELD EMISSION GUN
Accelerating Voltage300 kV
Electron Dose25 e/A**2
Illumination ModeFLOOD BEAM
Lens
MagnificationNominal: 40000, Calibrated: 38500
AstigmatismObjective lens astigmatism was corrected based on appearance of carbon film at 250,000 times magnification
Nominal Cs1.6 mm
Imaging ModeBRIGHT FIELD
Defocus900 nm - 2000 nm
Specimen Holder
HolderTop-entry holder for liquid helium cooled stage (the temperature of the specimen in this holder is usually at 4K)
ModelOTHER
Temperature10 K ( 10 - 20 K)
Camera
DetectorKODAK SO-163 FILM
Image Acquisition
#1
Od Range1
ScannerOTHER
Number of Digital Images111
Sampling Size2.5
Quant Bit Number16
DetailsAll images recorded on film, developed in Kodak d19 developer
Processing
Methodhelical reconstruction
3D reconstruction
Resolution MethodFSC 0.5
DetailsFinal maps were calculated from 111 tube images(closed class) and 123 tube images (open class)
SoftwareMRC, and, own, programs
AlgorithmStandard Fourier-Bessel synthesis
CTF CorrectionEach tube image
Resolution By Author6.2 A
Helical
DetailsAlignment 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
Atomic Model Fitting
Model #0
DetailsProtocol: 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
Refinement Protocolflexible
Refinement SpaceREAL
SoftwareDireX
PDB Entry ID2BG9
PDB Chain IDA, B, C, D, E
Fitted Coordinate
PDB entry ID
Download
Data from EMDB
Header (meta data in XML format)emd-2071.xml (10 KB)
Map dataemd_2071.map.gz (2.6 MB)
Imagesemd-2071.tif (185.3 KB)
FTP directoryftp://ftp.pdbj.org/pub/emdb/structures/EMD-2071
Movie files
movie #1
.mp4 (H.264/MPEG-4 AVC format), 3.2 MB
.webm (WebM/VP8 format), 5 MB
Session file for UCSF-Chimera, 26.1 KB
movie #2
.mp4 (H.264/MPEG-4 AVC format), 3.1 MB
.webm (WebM/VP8 format), 4.4 MB
Session file for UCSF-Chimera, 26.2 KB
movie #3
.mp4 (H.264/MPEG-4 AVC format), 3.6 MB
.webm (WebM/VP8 format), 4.9 MB
Session file for UCSF-Chimera, 2.8 MB
movie #4
.mp4 (H.264/MPEG-4 AVC format), 3.5 MB
.webm (WebM/VP8 format), 4.6 MB