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- PDB-8qqm: nicotinic acetylcholine receptor in intact synaptic membrane -

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Basic information

Entry
Database: PDB / ID: 8qqm
Titlenicotinic acetylcholine receptor in intact synaptic membrane
Components
  • Acetylcholine receptor subunit alpha
  • Acetylcholine receptor subunit beta
  • Acetylcholine receptor subunit delta
  • Acetylcholine receptor subunit gamma
KeywordsMEMBRANE PROTEIN / Ion channel
Function / homology
Function and homology information


acetylcholine-gated monoatomic cation-selective channel activity / 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 beta / Acetylcholine receptor subunit gamma / Acetylcholine receptor subunit delta
Similarity search - Component
Biological speciesTetronarce californica (Pacific electric ray)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.7 Å
AuthorsUnwin, N.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
UK Research and Innovation (UKRI)MC_U105184294 United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Influence of lipid bilayer on the structure of the muscle-type nicotinic acetylcholine receptor.
Authors: Nigel Unwin /
Abstract: The muscle-type nicotinic acetylcholine receptor is a transmitter-gated ion channel residing in the plasma membrane of electrocytes and striated muscle cells. It is present predominantly at synaptic ...The muscle-type nicotinic acetylcholine receptor is a transmitter-gated ion channel residing in the plasma membrane of electrocytes and striated muscle cells. It is present predominantly at synaptic junctions, where it effects rapid depolarization of the postsynaptic membrane in response to acetylcholine released into the synaptic cleft. Previously, cryo-EM of intact membrane from revealed that the lipid bilayer surrounding the junctional receptor has a uniquely asymmetric and ordered structure, due to a high concentration of cholesterol. It is now shown that this special lipid environment influences the transmembrane (TM) folding of the protein. All five submembrane MX helices of the membrane-intact junctional receptor align parallel to the surface of the cholesterol-ordered lipids in the inner leaflet of the bilayer; also, the TM helices in the outer leaflet are splayed apart. However in the structure obtained from the same protein after extraction and incorporation in nanodiscs, the MX helices do not align to a planar surface, and the TM helices arrange compactly in the outer leaflet. Realignment of the MX helices of the nanodisc-solved structure to a planar surface converts their adjoining TM helices into an obligatory splayed configuration, characteristic of the junctional receptor. Thus, the form of the receptor sustained by the special lipid environment of the synaptic junction is the one that mediates fast synaptic transmission; whereas, the nanodisc-embedded protein may be like the extrajunctional form, existing in a disordered lipid environment.
History
DepositionOct 5, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 8, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Acetylcholine receptor subunit alpha
B: Acetylcholine receptor subunit delta
C: Acetylcholine receptor subunit beta
D: Acetylcholine receptor subunit alpha
E: Acetylcholine receptor subunit gamma


Theoretical massNumber of molelcules
Total (without water)268,0295
Polymers268,0295
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area16080 Å2
ΔGint-163 kcal/mol
Surface area45870 Å2

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Components

#1: Protein Acetylcholine receptor subunit alpha /


Mass: 50168.164 Da / Num. of mol.: 2 / Source method: isolated from a natural source
Details: The extracellular domain is excluded from this analysis
Source: (natural) Tetronarce californica (Pacific electric ray)
References: UniProt: P02710
#2: Protein Acetylcholine receptor subunit delta /


Mass: 57625.711 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Details: The extracellular domain is excluded from this analysis
Source: (natural) Tetronarce californica (Pacific electric ray)
References: UniProt: P02718
#3: Protein Acetylcholine receptor subunit beta /


Mass: 53731.773 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Details: The extracellular domain is excluded from this analysis
Source: (natural) Tetronarce californica (Pacific electric ray)
References: UniProt: P02712
#4: Protein Acetylcholine receptor subunit gamma /


Mass: 56335.684 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Details: The extracellular domain is excluded from this analysis
Source: (natural) Tetronarce californica (Pacific electric ray)
References: UniProt: P02714

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Muscle-type nicotinic acetylcholine receptor in intact synaptic membrane
Type: ORGANELLE OR CELLULAR COMPONENT
Details: Postsynaptic membranes were isolated from fresh electric organ and incubated in low salt buffer to form ordered arrays of acetylcholine receptors in tubular vesicles
Entity ID: all / Source: NATURAL
Molecular weightValue: 0.29 MDa
Source (natural)Organism: Torpedo marmorata (marbled electric ray)
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMsodium cacodylateC2H7AsO21
21 mMcalcium chlorideCaCl21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Specimen comprises tubular vesicles which are imaged in thin ice over holes in the support film
Specimen supportDetails: Grids were washed extensively in chloroform and coated with additional carbon before use
Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 283 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: All images taken manually: by searching at low magnification for long straight and narrow tubes, then recording in integrating mode
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 59000 X / Calibrated magnification: 104478 X / Nominal defocus max: 2800 nm / Nominal defocus min: 1200 nm / Calibrated defocus min: 1200 nm / Calibrated defocus max: 2800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K
Image recordingAverage exposure time: 2 sec. / Electron dose: 40 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 200 / Num. of real images: 4045
Details: Images were collected in integrating mode, 2 seconds exposure

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Processing

EM software
IDNameVersionCategory
1RELION2.1 and 3.1particle selection
2DigitalMicrographimage acquisition
4GctfCTF correction
7DireXmodel fitting
11RELION2.1classification
12RELION2.13D reconstruction
13DireXmodel refinement
Image processingDetails: Images of appropriate helical families were selected on the basis of their FFTs, then drift-corrected and dose-weighted
CTF correctionDetails: Correction performed by standard procedure in RELION
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 160652
Details: 160652 tube segments from 4045 selected micrographs were reduced to 107524 tube segments after 2D classification
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 107524 / Algorithm: FOURIER SPACE
Details: The volumes for FSC determination were cut out from helical reconstructions of each class average
Num. of class averages: 34 / Symmetry type: POINT
Atomic model buildingB value: 350 / Protocol: FLEXIBLE FIT / Space: REAL
Details: Refinement parameters were chosen to minimise changes to the original secondary structure
Atomic model building

3D fitting-ID: 1 / Accession code: 7SMM / Details: initial model is of complete protein, PDB entry 7smm / Initial refinement model-ID: 1 / PDB-ID: 7SMM

7smm

/ Source name: PDB / Type: experimental model

IDPdb chain-IDChain-IDChain residue rangePdb chain residue range
1AA212-433212-433
2BB226-479226-479
3CC218-460218-460
4DD212-433212-433
5EE220-472220-472

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