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- EMDB-2382: The structure and super-organization of acetylcholine receptor-ra... -

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Basic information

Entry
Database: EMDB / ID: EMD-2382
TitleThe structure and super-organization of acetylcholine receptor-rapsyn complexes; class D
Map dataAcetylcholine receptor with two pairs of rapsyn bound at approximately 180 degrees apart
Sample
  • Sample: Postsynaptic membrane isolated from Torpedo marmorata electric organ
  • Protein or peptide: nicotinic acetylcholine receptor
  • Protein or peptide: Rapsyn
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 / neuron projection / synapse / zinc ion binding
Similarity search - Function
43kDa postsynaptic protein / Tetratricopeptide repeat 1 / 43kDa postsynaptic, conserved site / Rapsyn, myristoylation/linker region, N-terminal / : / Nicotinic acetylcholine receptor / Nicotinic acetylcholine receptor / Neurotransmitter-gated ion-channel, conserved site / Neurotransmitter-gated ion-channels signature. / Neurotransmitter-gated ion-channel transmembrane domain ...43kDa postsynaptic protein / Tetratricopeptide repeat 1 / 43kDa postsynaptic, conserved site / Rapsyn, myristoylation/linker region, N-terminal / : / Nicotinic acetylcholine receptor / 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 / 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: 42.0 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 22, 2013-
Header (metadata) releaseMay 29, 2013-
Map releaseJun 26, 2013-
UpdateJul 3, 2013-
Current statusJul 3, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.278
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.278
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4bor
  • Surface level: 0.278
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2382.tif

Downloads & links

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Map

FileDownload / File: emd_2382.map.gz / Format: CCP4 / Size: 825.2 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAcetylcholine receptor with two pairs of rapsyn bound at approximately 180 degrees apart
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
7.48 Å/pix.
x 60 pix.
= 448.8 Å		7.48 Å/pix.
x 60 pix.
= 448.8 Å		7.48 Å/pix.
x 60 pix.
= 448.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 7.48 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.278 / Movie #1: 0.278
Minimum - Maximum-0.77097201 - 1.1117115
Average (Standard dev.)0.0 (±0.06677666)
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
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS606060
D min/max/mean-0.7711.1120.000

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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: Rapsyn

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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 two pairs of rapsyns
Number unique components: 2
Molecular weightTheoretical: 474 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 densities
Number of copies: 4 / 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 discharged 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
TemperatureMin: 80 K / Max: 95 K / Average: 85 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 69,000 times magnification
Specialist opticsEnergy filter - Name: Gatan / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 80213 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 6.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 69000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN / Tilt series - Axis1 - Min angle: -70 ° / Tilt series - Axis1 - Max angle: 70 °
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing #1

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

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Image processing #2

Image processing ID2
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 42.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: XMIPP, ML, TOMO / Number subtomograms used: 3564
Final 3D classificationNumber classes: 5

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

PDB-4bor:
The structure and super-organization of acetylcholine receptor-rapsyn complexes class D

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