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- EMDB-10747: AP2 core in physiological buffer -

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Basic information

Entry
Database: EMDB / ID: EMD-10747
TitleAP2 core in physiological buffer
Map data
SampleAP2 core:
AP-2 complex subunits alpha and mu / (AP-2 complex subunit ...) x 6
Function / homology
Function and homology information


regulation of vesicle size / clathrin adaptor complex / postsynaptic neurotransmitter receptor internalization / AP-2 adaptor complex / low-density lipoprotein particle receptor catabolic process / positive regulation of protein localization to membrane / clathrin adaptor activity / coronary vasculature development / positive regulation of synaptic vesicle endocytosis / vesicle budding from membrane ...regulation of vesicle size / clathrin adaptor complex / postsynaptic neurotransmitter receptor internalization / AP-2 adaptor complex / low-density lipoprotein particle receptor catabolic process / positive regulation of protein localization to membrane / clathrin adaptor activity / coronary vasculature development / positive regulation of synaptic vesicle endocytosis / vesicle budding from membrane / neurotransmitter receptor internalization / endolysosome membrane / signal sequence binding / clathrin coat assembly / clathrin-dependent endocytosis / clathrin-coated endocytic vesicle / low-density lipoprotein particle clearance / aorta development / negative regulation of protein localization to plasma membrane / positive regulation of endocytosis / clathrin binding / ventricular septum development / clathrin-coated endocytic vesicle membrane / synaptic vesicle endocytosis / positive regulation of receptor internalization / low-density lipoprotein particle receptor binding / Wnt signaling pathway, planar cell polarity pathway / clathrin-coated pit / antigen processing and presentation of exogenous peptide antigen via MHC class II / vesicle-mediated transport / receptor internalization / negative regulation of neuron death / intracellular protein transport / ephrin receptor signaling pathway / terminal bouton / endocytic vesicle membrane / regulation of defense response to virus by virus / postsynapse / endocytosis / kidney development / protein-containing complex assembly / disordered domain specific binding / membrane organization / ion channel binding / cytoplasmic vesicle / lipid binding / glutamatergic synapse / intracellular membrane-bounded organelle / protein-containing complex binding / protein kinase binding / membrane / plasma membrane / cytosol
Clathrin adaptor, appendage, Ig-like subdomain superfamily / Clathrin adaptor, alpha/beta/gamma-adaptin, appendage, Ig-like subdomain / Clathrin adaptor, mu subunit, conserved site / AP-1/2/4 complex subunit beta / Clathrin adaptor complex, small chain / AP complex subunit beta / Clathrin adaptor, mu subunit / Clathrin/coatomer adaptor, adaptin-like, N-terminal / Clathrin adaptor, alpha-adaptin, appendage, C-terminal subdomain / Adaptor protein complex AP-2, alpha subunit ...Clathrin adaptor, appendage, Ig-like subdomain superfamily / Clathrin adaptor, alpha/beta/gamma-adaptin, appendage, Ig-like subdomain / Clathrin adaptor, mu subunit, conserved site / AP-1/2/4 complex subunit beta / Clathrin adaptor complex, small chain / AP complex subunit beta / Clathrin adaptor, mu subunit / Clathrin/coatomer adaptor, adaptin-like, N-terminal / Clathrin adaptor, alpha-adaptin, appendage, C-terminal subdomain / Adaptor protein complex AP-2, alpha subunit / Beta-adaptin appendage, C-terminal subdomain / AP-2 complex subunit sigma / Coatomer/calthrin adaptor appendage, C-terminal subdomain / Armadillo / Mu homology domain / AP-2 complex subunit mu, C-terminal superfamily / Armadillo-type fold / Longin-like domain superfamily / Armadillo-like helical / TBP domain superfamily / Mu2, C-terminal domain / AP-2 complex subunit mu, N-terminal / Adaptor protein complex, sigma subunit / Clathrin adaptor, beta-adaptin, appendage, Ig-like subdomain / AP complex, mu/sigma subunit
AP-2 complex subunit sigma / AP-2 complex subunit beta / AP-2 complex subunit mu / AP-2 complex subunit alpha
Biological speciesRattus norvegicus (Norway rat) / Homo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsKane Dickson V / Kovtun O / Kelly BT / Owen DJ / Briggs JAG
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Wellcome Trust207455/Z/17/Z United Kingdom
Medical Research Council (MRC, United Kingdom)MC_UP_1201/16 United Kingdom
CitationJournal: Sci Adv / Year: 2020
Title: Architecture of the AP2/clathrin coat on the membranes of clathrin-coated vesicles.
Authors: Oleksiy Kovtun / Veronica Kane Dickson / Bernard T Kelly / David J Owen / John A G Briggs /
Abstract: Clathrin-mediated endocytosis (CME) is crucial for modulating the protein composition of a cell's plasma membrane. Clathrin forms a cage-like, polyhedral outer scaffold around a vesicle, to which ...Clathrin-mediated endocytosis (CME) is crucial for modulating the protein composition of a cell's plasma membrane. Clathrin forms a cage-like, polyhedral outer scaffold around a vesicle, to which cargo-selecting clathrin adaptors are attached. Adaptor protein complex (AP2) is the key adaptor in CME. Crystallography has shown AP2 to adopt a range of conformations. Here, we used cryo-electron microscopy, tomography, and subtomogram averaging to determine structures, interactions, and arrangements of clathrin and AP2 at the key steps of coat assembly, from AP2 in solution to membrane-assembled clathrin-coated vesicles (CCVs). AP2 binds cargo and PtdIns(4,5) (phosphatidylinositol 4,5-bisphosphate)-containing membranes via multiple interfaces, undergoing conformational rearrangement from its cytosolic state. The binding mode of AP2 β2 appendage into the clathrin lattice in CCVs and buds implies how the adaptor structurally modulates coat curvature and coat disassembly.
Validation ReportPDB-ID: 6yae

SummaryFull reportAbout validation report
History
DepositionMar 12, 2020-
Header (metadata) releaseJul 29, 2020-
Map releaseJul 29, 2020-
UpdateAug 19, 2020-
Current statusAug 19, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.9
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.9
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6yae
  • Surface level: 0.9
  • 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_10747.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 240 pix.
= 256.8 Å
1.07 Å/pix.
x 240 pix.
= 256.8 Å
1.07 Å/pix.
x 240 pix.
= 256.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.9 / Movie #1: 0.9
Minimum - Maximum-2.2413461 - 4.016664
Average (Standard dev.)0.0014063013 (±0.15118398)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 256.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z256.800256.800256.800
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ280280280
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-2.2414.0170.001

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Supplemental data

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Segmentation: #1

Fileemd_10747_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #1

Fileemd_10747_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #2

Fileemd_10747_half_map_2.map
Projections & Slices
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Density Histograms

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Sample components

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Entire AP2 core

EntireName: AP2 core / Number of components: 8

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Component #1: protein, AP2 core

ProteinName: AP2 core / Recombinant expression: No
MassTheoretical: 270 kDa

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Component #2: protein, AP-2 complex subunits alpha and mu

ProteinName: AP-2 complex subunits alpha and mu / Recombinant expression: No
SourceSpecies: Rattus norvegicus (Norway rat)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: protein, AP-2 complex subunit beta

ProteinName: AP-2 complex subunit beta / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: protein, AP-2 complex subunit sigma

ProteinName: AP-2 complex subunit sigma / Recombinant expression: No
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #5: protein, AP-2 complex subunit alpha

ProteinName: AP-2 complex subunit alpha / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 69.656297 kDa
SourceSpecies: Rattus norvegicus (Norway rat)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #6: protein, AP-2 complex subunit beta

ProteinName: AP-2 complex subunit beta / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 66.953195 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #7: protein, AP-2 complex subunit mu

ProteinName: AP-2 complex subunit mu / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 49.726641 kDa
SourceSpecies: Rattus norvegicus (Norway rat)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #8: protein, AP-2 complex subunit sigma

ProteinName: AP-2 complex subunit sigma / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 17.038688 kDa
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.5 mg/mL / pH: 7.2
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 32.4 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: FEI FALCON III (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 105000
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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