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Gating movement in acetylcholine receptor analysed by time-resolved electron cryo-microscopy
by helical reconstruction, at 6.2 A resolution
#1: Surface view with section colored by density value, Surface level: 1.2, Made by UCSF CHIMERA
#2: Surface view colored by cylindrical radius, Surface level: 1.2, Made by UCSF CHIMERA
#3: Surface view with fitted model, atomic models: PDB-4aq5, Surface level: 1.2, Made by UCSF CHIMERA
#4: Simplified surface model with fitted atomic model: PDB-4aq5, Made by Jmol
 Entry | |
| Summary | |
| Database / ID | EM DATA BANK (EMDB) / 2071 |
|---|---|
| Authors | Unwin N, Fujiyoshi Y |
| EMDB Sites |  EMDB @PDBe (EU), EMDB @RCSB (USA) |
| Structure Visualization | |
| Movies |  Movie Page#1: Surface view with section colored by density value, Surface level: 1.2, Made by UCSF CHIMERA #2: Surface view colored by cylindrical radius, Surface level: 1.2, Made by UCSF CHIMERA #3: Surface view with fitted model, atomic models: PDB-4aq5, Surface level: 1.2, Made by UCSF CHIMERA #4: Simplified surface model with fitted atomic model: PDB-4aq5, Made by Jmol |
| Supplemental images | |
| Structure viewers |  Yorodumi,  Launch PeppeR (About PeppeR), Volume viewer (RCSB, PDBe) |
| Related Structure Data | |
| Related Entries |
Cite: data citing same article Fit: output model of fitting |
| Similar strucutres (beta) |
 List of similar structure data  about Omokage system |
 Article | |
| Citation - Primary | |
| Article | J. Mol. Biol., Vol. 422, Issue 5, Page 617-34, Year 2012 |
|---|---|
| Title | Gating movement of acetylcholine receptor caught by plunge-freezing. |
| Authors | Nigel Unwin, Yoshinori Fujiyoshi MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK. |
| Keywords |  Acetylcholine (metabolism, 51-84-3), Allosteric Regulation, Animals, Cryoelectron Microscopy (methods), Ion Channel Gating, Models, Biological, Models, Molecular, Protein Conformation, Receptors, Cholinergic (chemistry), Torpedo |
| Links | PII: S0022-2836(12)00577-3, DOI: 10.1016/j.jmb.2012.07.010, PubMed: 22841691, PMC: PMC3443390 |
 Map | |||||||||||||||||||||||||
| File |  EMD-2071.map ( map file in CCP4 format, 11012 KB ) | ||||||||||||||||||||||||
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| Projections & Slices | Size of images:
Images are generated by Spider package. | ||||||||||||||||||||||||
| Density |
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| Data Type | Image stored as Reals | ||||||||||||||||||||||||
| Space Group Number | 1 | ||||||||||||||||||||||||
| Map Geometry |
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| Unit Cell | A = 128.0 A , B = 128.0 A , C = 168.0 A , alpha = 90.0 degrees , beta = 90.0 degrees , gamma = 90.0 degrees | ||||||||||||||||||||||||
| Pixel Spacing | X = 1 A , Y = 1 A , Z = 1 A | ||||||||||||||||||||||||
| CCP4 map header info | |||||||||||||||||||||||||
| Annotation Details | Density map of acetylcholine receptor | ||||||||||||||||||||||||
 Supplement | |
| Images | |
| Images | |
|---|---|
 Sample | |
| Name | nicotinic acetylcholine receptor in native postsynaptic membrane from Torpedo marmorata |
|---|---|
| Number of Components | 1 |
| Oligomeric State | 5 subunits |
| Theoretical Mass | 0.3 MDa |
| Mass-estimation Method | molecular weight based on amino acid sequence data and attached sugars |
| Experimental Mass | 0.3 MDa |
| Component #1: protein - nicotinic receptor | |
| Scientific name | nicotinic acetylcholine receptor |
| Common Name | nicotinic receptor |
| Theoretical Mass | 0.3 MDa |
| Experimental Mass | 0.3 MDa |
| Details | Protein is embedded in postsynaptic membrane isolated from Torpedo marmorata electric organ |
| Oligomeric Details | pentamer |
| Scientific Name of Species | Torpedo marmorata (NCBI Taxonomy: 7788) |
| Common Name of Species | marbled electric ray |
| Recombinant expression | No |
| Natural Source | Cell: electrocyte cells Organ Or Tissue: electric organ Organelle: plasma membrane Cell Location: plasma membrane |
 Experiment | |
| Sample Preparation | |
| Helical Parameters | Hand: RIGHT HANDED |
|---|---|
| Specimen Support Details | 300 mesh copper grid with pre-irradiated thick holey carbon support, glow discharged in amylamine atmosphere |
| Specimen State | helicalArray |
| Crystal Grow Details | Tubular membrane crystals of acetylcholine receptors grow spontaneously from isolated postsynaptic membranes when incubated in low salt buffer at 17 degrees C for two weeks |
| Buffer | pH: 7.0 Details: 100 mM sodium cacodylate, 1 mM calcium chloride |
| Vitrification | |
| Cryogen Name | ETHANE |
| Method | Blot until applied droplet loses contact with filter paper (indicated by loss of transparency; typically 6s) |
| Temperature | 120 Kelvin |
| 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 |
| Instrument | HOMEMADE PLUNGER |
| Humidity | 85 |
| Imaging | |
| Microscope | JEOL 3000SFF |
| Date | 01-NOV-2005 |
| Details | Standard low dose imaging of specimens over holes in the carbon support film |
| Electron Gun | |
| Electron Source | FIELD EMISSION GUN |
| Accelerating Voltage | 300 kV |
| Electron Dose | 25 e/A**2 |
| Illumination Mode | FLOOD BEAM |
| Lens | |
| Magnification | Nominal: 40000 X, Calibrated: 38500 X |
| Astigmatism | Objective lens astigmatism was corrected based on appearance of carbon film at 250,000 times magnification |
| Nominal Cs | 1.6 mm |
| Imaging Mode | BRIGHT FIELD |
| Defocus | 900 nm - 2000 nm |
| Specimen Holder | |
| Holder | Top-entry holder for liquid helium cooled stage (the temperature of the specimen in this holder is usually at 4K) ( OTHER ) |
| Temperature | 10 Kelvin ( 10 Kelvin - 20 Kelvin ) |
| Camera | |
| Detector | Kodak SO163 film |
| Image Acquisition | |
| Od Range | 1.0 |
| Scanner | OTHER |
| Number of Digital Images | 111 |
| Sampling Size | 2.5 microns |
| Quant Bit Number | 16 |
| Details | All images recorded on film, developed in Kodak d19 developer |
 Processing | |
| Method | helical reconstruction |
|---|---|
| 3 D reconstruction | |
| Resolution Method | FSC at 0.5 cut-off |
| 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 By Author | 6.2 |
| Helical | |
| 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 |
| Atomic Model Fitting | |
| Model #0 | |
| 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 |
| Refinement Protocol | flexible |
| Refinement Space | REAL |
| Software | DireX |
| Fitted Coordinate | |
| PDB entry ID | |
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