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

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Basic information

Entry
Database: EMDB / ID: EMD-2071
TitleGating movement in acetylcholine receptor analysed by time-resolved electron cryo-microscopy
Map dataDensity map of acetylcholine receptor
Sample
  • Sample: nicotinic acetylcholine receptor in native postsynaptic membrane from Torpedo marmorata
  • Protein or peptide: nicotinic acetylcholine receptor
Keywordsacetylcholine receptor / freeze-trapping / asymmetric gating / allosteric mechanism
Function / homology
Function and homology information


acetylcholine-gated channel complex / acetylcholine-gated monoatomic cation-selective channel activity / acetylcholine receptor signaling pathway / transmembrane signaling receptor activity / postsynaptic membrane / neuron projection
Similarity search - Function
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
Similarity search - Domain/homology
Acetylcholine receptor subunit alpha / Acetylcholine receptor subunit delta / Acetylcholine receptor gamma subunit / Acetylcholine receptor subunit beta
Similarity search - Component
Biological speciesTorpedo marmorata (marbled electric ray)
Methodhelical reconstruction / cryo EM / Resolution: 6.2 Å
AuthorsUnwin N / Fujiyoshi Y
CitationJournal: J Mol Biol / Year: 2012
Title: Gating movement of acetylcholine receptor caught by plunge-freezing.
Authors: Nigel Unwin / Yoshinori Fujiyoshi /
Abstract: The nicotinic acetylcholine (ACh) receptor converts transiently to an open-channel form when activated by ACh released into the synaptic cleft. We describe here the conformational change underlying ...The nicotinic acetylcholine (ACh) receptor converts transiently to an open-channel form when activated by ACh released into the synaptic cleft. We describe here the conformational change underlying this event, determined by electron microscopy of ACh-sprayed and freeze-trapped postsynaptic membranes. ACh binding to the α subunits triggers a concerted rearrangement in the ligand-binding domain, involving an ~1-Å outward displacement of the extracellular portion of the β subunit where it interacts with the juxtaposed ends of α-helices shaping the narrow membrane-spanning pore. The β-subunit helices tilt outward to accommodate this displacement, destabilising the arrangement of pore-lining helices, which in the closed channel bend inward symmetrically to form a central hydrophobic gate. Straightening and tangential motion of the pore-lining helices effect channel opening by widening the pore asymmetrically and increasing its polarity in the region of the gate. The pore-lining helices of the α(γ) and δ subunits, by flexing between alternative bent and straight conformations, undergo the greatest movements. This coupled allosteric transition shifts the structure from a tense (closed) state toward a more relaxed (open) state.
History
DepositionApr 12, 2012-
Header (metadata) releaseApr 17, 2012-
Map releaseAug 1, 2012-
UpdateSep 26, 2012-
Current statusSep 26, 2012Processing site: PDBe / Status: Released

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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 modelsPDB-4aq5
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd-2071.tif

Downloads & links

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Map

FileDownload / File: emd_2071.map.gz / Format: CCP4 / Size: 10.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDensity map of acetylcholine receptor
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
1 Å/pix.
x 168 pix.
= 168. Å		1 Å/pix.
x 128 pix.
= 128. Å		1 Å/pix.
x 128 pix.
= 128. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.2 / Movie #1: 1.2
Minimum - Maximum-3.90023351 - 4.90560436
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin000
Dimensions128128168
Spacing128128168
CellA: 128.0 Å / B: 128.0 Å / C: 168.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z111
M x/y/z128128168
origin x/y/z0.0000.0000.000
length x/y/z128.000128.000168.000
α/β/γ90.00090.00090.000
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
Components
  • Sample: nicotinic acetylcholine receptor in native postsynaptic membrane from Torpedo marmorata
  • Protein or peptide: nicotinic acetylcholine receptor

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Supramolecule #1000: nicotinic acetylcholine receptor in native postsynaptic membrane ...

SupramoleculeName: nicotinic acetylcholine receptor in native postsynaptic membrane from Torpedo marmorata
type: sample / ID: 1000 / Oligomeric state: 5 subunits / Number unique components: 1
Molecular weightExperimental: 300 KDa / Theoretical: 300 KDa
Method: molecular weight based on amino acid sequence data and attached sugars

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Macromolecule #1: nicotinic acetylcholine receptor

MacromoleculeName: nicotinic acetylcholine receptor / type: protein_or_peptide / ID: 1 / Name.synonym: nicotinic receptor
Details: Protein is embedded in postsynaptic membrane isolated from Torpedo marmorata electric organ
Oligomeric state: pentamer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Torpedo marmorata (marbled electric ray) / synonym: marbled electric ray / Tissue: electric organ / Cell: electrocyte cells / Organelle: plasma membrane / Location in cell: plasma membrane
Molecular weightExperimental: 300 KDa / Theoretical: 300 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

BufferpH: 7 / Details: 100 mM sodium cacodylate, 1 mM calcium chloride
GridDetails: 300 mesh copper grid with pre-irradiated thick holey carbon support, glow discharged in amylamine atmosphere
VitrificationCryogen name: ETHANE / Chamber humidity: 85 % / Chamber temperature: 120 K / Instrument: HOMEMADE PLUNGER
Details: Vitrification carried out at an ambient temperature of 8 degrees C
Timed 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)
Method: Blot until applied droplet loses contact with filter paper (indicated by loss of transparency; typically 6s)
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

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Electron microscopy

MicroscopeJEOL 3000SFF
TemperatureMin: 10 K / Max: 20 K / Average: 10 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected based on appearance of carbon film at 250,000 times magnification
DetailsStandard low dose imaging of specimens over holes in the carbon support film
DateNov 1, 2005
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 2.5 µm / Number real images: 111 / Average electron dose: 25 e/Å2
Details: All images recorded on film, developed in Kodak d19 developer
Od range: 1 / Bits/pixel: 16
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 38500 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 1.6 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 40000
Sample stageSpecimen holder: Top-entry holder for liquid helium cooled stage (the temperature of the specimen in this holder is usually at 4K)
Specimen holder model: OTHER

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Image processing

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
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 6.2 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: MRC, and, own, programs
Details: Final maps were calculated from 111 tube images(closed class) and 123 tube images (open class)
CTF correctionDetails: Each tube image

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Atomic model buiding 1

Initial modelPDB ID:

2bg9


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D / Chain - #4 - Chain ID: E
SoftwareName: DireX
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
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-4aq5:
Gating movement in acetylcholine receptor analysed by time-resolved electron cryo-microscopy (closed class)

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