EMDB-1044: Structure and gating mechanism of the acetylcholine receptor pore.

Basic information

• Entry: Database: EMDB / ID: EMD-1044
• Title: Structure and gating mechanism of the acetylcholine receptor pore.
• Map data: The map is of the membrane-spanning domain of the nicotinic acetylcholine receptor in the closed state, viewed from the synaptic cleft. The arrangement of subunits around the central axis, clockwise beginning from the bottom (closest to 0 on the y-axis) ia alpha, gamma, beta, delta. The Fourier terms were derived from tubular crystals having helical symmetry. They are of higher quality along the meridional (y-axis) direction than the equatorial direction (where the diffraction is weaker and there is additional noise associated with layer-line overlap. This has resulted in some asymmetry in the map, with the best direction being along the axis of the tube (y-axis). The map was obtained by averaging data from four helical families in real space, weighting each family approximately according to the number of receptors analysed. The actual weights were: 0.70 (-16,6); 0.30 (-15,7); 0.30 (-17,5); 0.25 (-18,6). As explained in the Reference, the dominating low resolution terms were weakened by subtracting a map of the structure with terms extending to only 15 Angstroms. THe weight used for the subtraction map was -0.88. The terms along the equator have also been included with a weight of 0.04, so that the densities corresponding to the alpha-helical segments are represented at about the same level throughout the thickness of the bilayer.

Sample

• Sample: Crystalline postsynaptic membrane from Torpedo marmorata electric organ
• Organelle or cellular component: postsynaptic membrane lipids
• Protein or peptide: acetylcholine receptor

Function / homology

Function:

acetylcholine-gated channel complex / acetylcholine-gated monoatomic cation-selective channel activity / acetylcholine receptor signaling pathway / transmembrane signaling receptor activity / postsynaptic membrane

Domain/homology:

Nicotinic acetylcholine receptor / Neurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channels signature. / Neurotransmitter-gated ion-channel transmembrane domain / Neurotransmitter-gated ion-channel transmembrane region / Neurotransmitter-gated ion-channel transmembrane domain superfamily / Neuronal acetylcholine receptor / Neurotransmitter-gated ion-channel / Neurotransmitter-gated ion-channel ligand-binding domain / Neurotransmitter-gated ion-channel ligand-binding domain superfamily / Neurotransmitter-gated ion-channel ligand binding domain

Component:

Acetylcholine receptor subunit alpha / Acetylcholine receptor subunit beta / Acetylcholine receptor subunit gamma / Acetylcholine receptor subunit delta / Acetylcholine receptor subunit delta / Acetylcholine receptor gamma subunit / Acetylcholine receptor subunit beta

• Biological species: Torpedo marmorata (marbled electric ray)
• Method: helical reconstruction / cryo EM / negative staining / Resolution: 4.0 Å
• Authors: Miyazawa A / Fujiyoshi Y / Unwin N
• Citation: Journal: Nature / Year: 2003; Title: Structure and gating mechanism of the acetylcholine receptor pore.; Authors: Atsuo Miyazawa / Yoshinori Fujiyoshi / Nigel Unwin / ; Abstract: The nicotinic acetylcholine receptor controls electrical signalling between nerve and muscle cells by opening and closing a gated, membrane-spanning pore. Here we present an atomic model of the closed pore, obtained by electron microscopy of crystalline postsynaptic membranes. The pore is shaped by an inner ring of 5 alpha-helices, which curve radially to create a tapering path for the ions, and an outer ring of 15 alpha-helices, which coil around each other and shield the inner ring from the lipids. The gate is a constricting hydrophobic girdle at the middle of the lipid bilayer, formed by weak interactions between neighbouring inner helices. When acetylcholine enters the ligand-binding domain, it triggers rotations of the protein chains on opposite sides of the entrance to the pore. These rotations are communicated through the inner helices, and open the pore by breaking the girdle apart.

History

Deposition	Apr 3, 2003	-
Header (metadata) release	Apr 9, 2003	-
Map release	Apr 9, 2005	-
Update	Jun 18, 2014	-
Current status	Jun 18, 2014	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_1044.map.gz / Format: CCP4 / Size: 3.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: The map is of the membrane-spanning domain of the nicotinic acetylcholine receptor in the closed state, viewed from the synaptic cleft. The arrangement of subunits around the central axis, clockwise beginning from the bottom (closest to 0 on the y-axis) ia alpha, gamma, beta, delta. The Fourier terms were derived from tubular crystals having helical symmetry. They are of higher quality along the meridional (y-axis) direction than the equatorial direction (where the diffraction is weaker and there is additional noise associated with layer-line overlap. This has resulted in some asymmetry in the map, with the best direction being along the axis of the tube (y-axis). The map was obtained by averaging data from four helical families in real space, weighting each family approximately according to the number of receptors analysed. The actual weights were: 0.70 (-16,6); 0.30 (-15,7); 0.30 (-17,5); 0.25 (-18,6). As explained in the Reference, the dominating low resolution terms were weakened by subtracting a map of the structure with terms extending to only 15 Angstroms. THe weight used for the subtraction map was -0.88. The terms along the equator have also been included with a weight of 0.04, so that the densities corresponding to the alpha-helical segments are represented at about the same level throughout the thickness of the bilayer.
• Voxel size: X=Y=Z: 1 Å

Density

Contour Level	1: 2.18 / Movie #1: 2.2
Minimum - Maximum	-2.43914 - 6.31988
Average (Standard dev.)	-0.000000169242 (±1.0)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order Y X Z Origin 0 0 52 Dimensions 128 128 55 Spacing 128 128 55
Cell	A: 128 Å / B: 128 Å / C: 168 Å α=β=γ: 90 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	1	1	1
M x/y/z	128	128	168
origin x/y/z	0.000	0.000	0.000
length x/y/z	128.000	128.000	168.000
α/β/γ	90.000	90.000	90.000
start NX/NY/NZ	0	0	52
NX/NY/NZ	128	128	55
MAP C/R/S	2	1	3
start NC/NR/NS	0	0	52
NC/NR/NS	128	128	55
D min/max/mean	-2.439	6.320	-0.000

Supplemental data

Sample components

Entire : Crystalline postsynaptic membrane from Torpedo marmorata electric...

• Entire: Name: Crystalline postsynaptic membrane from Torpedo marmorata electric organ

Components

• Sample: Crystalline postsynaptic membrane from Torpedo marmorata electric organ
• Organelle or cellular component: postsynaptic membrane lipids
• Protein or peptide: acetylcholine receptor

Supramolecule #1000: Crystalline postsynaptic membrane from Torpedo marmorata electric...

• Supramolecule: Name: Crystalline postsynaptic membrane from Torpedo marmorata electric organ; type: sample / ID: 1000 ; Oligomeric state: The acetylcholine receptors are hetero-pentamers composed of 2 alpha 1 beta 1 gamma and 1 delta subunit; Number unique components: 2

Supramolecule #1: postsynaptic membrane lipids

• Supramolecule: Name: postsynaptic membrane lipids / type: organelle_or_cellular_component / ID: 1 / Recombinant expression: No / Database: NCBI
• Source (natural): Organism: Torpedo marmorata (marbled electric ray) / synonym: Torpedo marmorata / Organelle: electric organ / Location in cell: plasma membrane

Macromolecule #1: acetylcholine receptor

• Macromolecule: Name: acetylcholine receptor / type: protein_or_peptide / ID: 1 ; Details: This is the MW of the glycosylated protein. The protein itself accounts for 258kD; Oligomeric state: pentamer / Recombinant expression: No / Database: NCBI
• Source (natural): Organism: Torpedo marmorata (marbled electric ray) / synonym: marbled electric ray / Organelle: electric organ / Location in cell: plasma membrane
• Molecular weight: Experimental: 290 KDa

Experimental details

Structure determination

• Method: negative staining, cryo EM
• Processing: helical reconstruction
• Aggregation state: filament

Sample preparation

• Buffer: pH: 6.8 / Details: 100mM sodium cacodylate, 1mM CaCl2
• Staining: Type: NEGATIVE / Details: no stains or fixatives used
• Grid: Details: holey carbon film made over 300 mesh copper grids. To minimise beam movement at the 4K imaging temperature, it was essential that the carbon films had a high electrical conductivity - achieved by evaporation of carbon in a high vacuum and pre-irradiation of the grids.
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 100 K / Instrument: HOMEMADE PLUNGER; Details: Vitrification instrument: Home-built model. The plunging apparatus was contained in a bench-top fridge having a window made in the door. Wet air was continually bubbled into the fridge, which was maintained at 4-8 deg. centigrade.; Method: The grid was first glow-discharged in the presence of amyl amine. The specimen was applied to the carbon-film side in 4.2ul droplets. Blotting was done from the other side, removing the filter paper and plunging as soon as the paper and grid were observed to lose water-contact with each other - typically after 6 seconds.

Electron microscopy

• Microscope: JEOL KYOTO-3000SFF
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Calibrated magnification: 36800 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 1.3 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 40000
• Sample stage: Specimen holder: top-entry / Specimen holder model: OTHER
• Temperature: Min: 4.2 K / Max: 4.2 K / Average: 4.2 K
• Alignment procedure: Legacy - Astigmatism: correction on carbon film at 250,000
• Image recording: Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 5 µm / Number real images: 359 / Average electron dose: 20 e/Ų; Details: Scanning done with a point-source, flat-bed Joyce-Loebl microdensitometer, modified in-house; Od range: 1 / Bits/pixel: 10

Image processing

• CTF correction: Details: Measurement of positions of Thon rings from area of tube that was processed
• Final reconstruction: Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: In-house software based on MRC system; Details: Layer-line data were collected from 4 helical families of tubes - (-16,6),(-15,7),(-17,5),(-18,6) - after dividing the tubes into short segments to correct for distortions. The maps calculated from each of the families were then averaged in real space to derive the final three-dimensional densities.
• Details: The specimens were tubular crystals forming a range of helical families, with the receptors in each case being organised on a p2 surface lattice.

Atomic model buiding 1

• Details: Interpretation of the experimental density map and model building into the densities were performed using O. The helical segments were fitted individually, using the protruding regions along the helical densities to identify the largest side chains. This allowed tentative assignments to be made of each amino acid according to the sequence, both along the helices and along the short connecting loops. These assignments were then validated for each subunit by checking their consistency with residues in equivalent positions around the pentamer.

Output model

PDB-1oed:
STRUCTURE OF ACETYLCHOLINE RECEPTOR PORE FROM ELECTRON IMAGES