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- EMDB-2377: The structure and super-organization of acetylcholine receptor-ra... -

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Basic information

Entry
Database: EMDB / ID: EMD-2377
TitleThe structure and super-organization of acetylcholine receptor-rapsyn complexes; class A
Map dataAcetylcholine receptor with one rapsyn bound
Sample
  • Sample: Postsynaptic membrane isolated from Torpedo marmorata electric organ
  • Protein or peptide: nicotinic acetylcholine receptor
  • Protein or peptide: RapsynRAPSN
Keywordsneurotransmitter receptor / clustering / synapse / neuromuscular junction / nicotinic / acetylcholine receptor / rapsyn / 43K / electric organ
Function / homology
Function and homology information


protein binding / acetylcholine-gated channel complex / acetylcholine-gated monoatomic cation-selective channel activity / acetylcholine receptor binding / acetylcholine receptor signaling pathway / transmembrane signaling receptor activity / cell junction / chemical synaptic transmission / postsynaptic membrane / cytoskeleton ...protein binding / acetylcholine-gated channel complex / acetylcholine-gated monoatomic cation-selective channel activity / acetylcholine receptor binding / acetylcholine receptor signaling pathway / transmembrane signaling receptor activity / cell junction / chemical synaptic transmission / postsynaptic membrane / cytoskeleton / synapse / zinc ion binding
Similarity search - Function
43kDa postsynaptic protein / 43kDa postsynaptic, conserved site / Rapsyn, myristoylation/linker region, N-terminal / : / Nicotinic acetylcholine receptor / Nicotinic acetylcholine receptor / Tetratricopeptide repeat 1 / Neurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channels signature. / Neurotransmitter-gated ion-channel transmembrane domain ...43kDa postsynaptic protein / 43kDa postsynaptic, conserved site / Rapsyn, myristoylation/linker region, N-terminal / : / Nicotinic acetylcholine receptor / Nicotinic acetylcholine receptor / Tetratricopeptide repeat 1 / 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 / Tetratricopeptide repeat / Zinc finger, RING-type / Tetratricopeptide-like helical domain superfamily / Zinc finger, RING/FYVE/PHD-type
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)
Methodsubtomogram averaging / cryo EM / negative staining / Resolution: 41.0 Å
AuthorsZuber B / Unwin N
CitationJournal: Proc Natl Acad Sci U S A / Year: 2013
Title: Structure and superorganization of acetylcholine receptor-rapsyn complexes.
Authors: Benoît Zuber / Nigel Unwin /
Abstract: The scaffolding protein at the neuromuscular junction, rapsyn, enables clustering of nicotinic acetylcholine receptors in high concentration and is critical for muscle function. Patients with ...The scaffolding protein at the neuromuscular junction, rapsyn, enables clustering of nicotinic acetylcholine receptors in high concentration and is critical for muscle function. Patients with insufficient receptor clustering suffer from muscle weakness. However, the detailed organization of the receptor-rapsyn network is poorly understood: it is unclear whether rapsyn first forms a wide meshwork to which receptors can subsequently dock or whether it only forms short bridges linking receptors together to make a large cluster. Furthermore, the number of rapsyn-binding sites per receptor (a heteropentamer) has been controversial. Here, we show by cryoelectron tomography and subtomogram averaging of Torpedo postsynaptic membrane that receptors are connected by up to three rapsyn bridges, the minimum number required to form a 2D network. Half of the receptors belong to rapsyn-connected groups comprising between two and fourteen receptors. Our results provide a structural basis for explaining the stability and low diffusion of receptors within clusters.
History
DepositionMay 20, 2013-
Header (metadata) releaseMay 29, 2013-
Map releaseJun 26, 2013-
UpdateAug 12, 2015-
Current statusAug 12, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.413
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.413
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4boi
  • Surface level: 0.413
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_2377.map.gz / Format: CCP4 / Size: 825.2 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAcetylcholine receptor with one rapsyn bound
Voxel sizeX=Y=Z: 7.48 Å
Density
Contour LevelBy AUTHOR: 0.413 / Movie #1: 0.413
Minimum - Maximum-0.68770933 - 1.22997284
Average (Standard dev.)0.0 (±0.07752144)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions606060
Spacing606060
CellA=B=C: 448.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z7.487.487.48
M x/y/z606060
origin x/y/z0.0000.0000.000
length x/y/z448.800448.800448.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS606060
D min/max/mean-0.6881.2300.000

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Supplemental data

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Sample components

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Entire : Postsynaptic membrane isolated from Torpedo marmorata electric organ

EntireName: Postsynaptic membrane isolated from Torpedo marmorata electric organ
Components
  • Sample: Postsynaptic membrane isolated from Torpedo marmorata electric organ
  • Protein or peptide: nicotinic acetylcholine receptor
  • Protein or peptide: RapsynRAPSN

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Supramolecule #1000: Postsynaptic membrane isolated from Torpedo marmorata electric organ

SupramoleculeName: Postsynaptic membrane isolated from Torpedo marmorata electric organ
type: sample / ID: 1000 / Details: class average
Oligomeric state: acetylcholine receptors bound to one rapsyn
Number unique components: 2
Molecular weightTheoretical: 336 KDa

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Macromolecule #1: nicotinic acetylcholine receptor

MacromoleculeName: nicotinic acetylcholine receptor / type: protein_or_peptide / ID: 1 / Name.synonym: AChR / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Torpedo marmorata (marbled electric ray) / synonym: Marble electric ray / Tissue: Electric organ / Cell: Electrocyte / Location in cell: Postsynaptic plasma membrane
Molecular weightExperimental: 290 KDa / Theoretical: 280 KDa
SequenceInterPro: Nicotinic acetylcholine receptor

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Macromolecule #2: Rapsyn

MacromoleculeName: Rapsyn / type: protein_or_peptide / ID: 2
Name.synonym: 43 kDa receptor-associated protein of the synapse
Details: the number of copies is suggested by the volume of the rapsyn density
Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Torpedo marmorata (marbled electric ray) / synonym: Marble electric ray / Tissue: Electric organ / Cell: Electrocyte / Location in cell: Postsynaptic plasma membrane
Molecular weightExperimental: 43 KDa / Theoretical: 46 KDa
SequenceGO: chemical synaptic transmission, protein binding, zinc ion binding, acetylcholine receptor binding, cytoskeleton, cell junction, synapse, postsynaptic membrane
InterPro: 43kDa postsynaptic protein, Rapsyn, myristoylation/linker region, N-terminal, Tetratricopeptide-like helical domain superfamily, INTERPRO: IPR013026, Zinc finger, RING/FYVE/PHD-type, Zinc ...InterPro: 43kDa postsynaptic protein, Rapsyn, myristoylation/linker region, N-terminal, Tetratricopeptide-like helical domain superfamily, INTERPRO: IPR013026, Zinc finger, RING/FYVE/PHD-type, Zinc finger, RING-type, Tetratricopeptide repeat, Tetratricopeptide repeat 1, 43kDa postsynaptic, conserved site

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
Details: 400 mM NaCl, 20 mM phosphate buffer, leupeptin 0.3 mg/l, pepstatin 1 mg/l
StainingType: NEGATIVE / Details: unstained
GridDetails: 300 mesh copper grid with lacy carbon support, glow discharge in amylamine atmosphere
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 78 K / Instrument: HOMEMADE PLUNGER / Method: blot from the carbon side

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 80213 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 6.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 69000
Specialist opticsEnergy filter - Name: Gatan / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN / Tilt series - Axis1 - Min angle: -70 ° / Tilt series - Axis1 - Max angle: 70 °
TemperatureMin: 80 K / Max: 95 K / Average: 85 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 69,000 times magnification
Detailsdual tilt axis tomography
DateJul 25, 2008
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 142 / Average electron dose: 50 e/Å2 / Details: dual axis tilt series / Bits/pixel: 16
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing #1

Final 3D classificationNumber classes: 5
Final reconstructionAlgorithm: OTHER / Software - Name: IMOD, PRIISM/IVE / Number subtomograms used: 3564
Image processing ID1

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Image processing #2

Final 3D classificationNumber classes: 5
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 41.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: XMIPP, ML, TOMO / Number subtomograms used: 3564
Image processing ID2

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Atomic model buiding 1

Initial modelPDB ID:

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: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-4boi:
The structure and super-organization of acetylcholine receptor-rapsyn complexes class A

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