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- EMDB-10749: AP2 dimer on a membrane containing tyrosine-based cargo peptide -

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Basic information

Entry
Database: EMDB / ID: EMD-10749
TitleAP2 dimer on a membrane containing tyrosine-based cargo peptide
Map dataSharpened map of AP2 dimer on membranes containing tyrosine-based cargo peptide
Sample
  • Complex: AP2 dimer on membrane
Function / homology
Function and homology information


protein metabolic process => GO:0019538 / Gap junction degradation / Formation of annular gap junctions / LDL clearance / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / LDL clearance / WNT5A-dependent internalization of FZD4 / VLDLR internalisation and degradation / Retrograde neurotrophin signalling ...protein metabolic process => GO:0019538 / Gap junction degradation / Formation of annular gap junctions / LDL clearance / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / LDL clearance / WNT5A-dependent internalization of FZD4 / VLDLR internalisation and degradation / Retrograde neurotrophin signalling / Nef Mediated CD8 Down-regulation / Trafficking of GluR2-containing AMPA receptors / Retrograde neurotrophin signalling / Retrograde transport at the Trans-Golgi-Network / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / clathrin adaptor complex / WNT5A-dependent internalization of FZD4 / Trafficking of GluR2-containing AMPA receptors / WNT5A-dependent internalization of FZD4 / extrinsic component of presynaptic endocytic zone membrane / VLDLR internalisation and degradation / MHC class II antigen presentation / Recycling pathway of L1 / AP-2 adaptor complex / regulation of vesicle size / postsynaptic neurotransmitter receptor internalization / Recycling pathway of L1 / Retrograde neurotrophin signalling / Cargo recognition for clathrin-mediated endocytosis / Cargo recognition for clathrin-mediated endocytosis / clathrin-coated endocytic vesicle / positive regulation of synaptic vesicle endocytosis / LDL clearance / Clathrin-mediated endocytosis / clathrin adaptor activity / Clathrin-mediated endocytosis / vesicle budding from membrane / membrane coat / membrane organization / trans-Golgi network transport vesicle / clathrin-dependent endocytosis / MHC class II antigen presentation / protein serine/threonine kinase binding / coronary vasculature development / signal sequence binding / endolysosome membrane / Nef Mediated CD4 Down-regulation / negative regulation of protein localization to plasma membrane / low-density lipoprotein particle receptor binding / aorta development / Neutrophil degranulation / ventricular septum development / clathrin binding / Golgi Associated Vesicle Biogenesis / Recycling pathway of L1 / Trafficking of GluR2-containing AMPA receptors / positive regulation of receptor internalization / synaptic vesicle endocytosis / EPH-ephrin mediated repulsion of cells / clathrin-coated pit / transport vesicle / vesicle-mediated transport / MHC class II antigen presentation / VLDLR internalisation and degradation / phosphatidylinositol binding / post-translational protein modification / kidney development / Post-translational protein phosphorylation / clathrin-coated endocytic vesicle membrane / intracellular protein transport / trans-Golgi network / terminal bouton / cytoplasmic side of plasma membrane / receptor internalization / kinase binding / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / endocytic vesicle membrane / disordered domain specific binding / synaptic vesicle / Cargo recognition for clathrin-mediated endocytosis / presynapse / Clathrin-mediated endocytosis / cytoplasmic vesicle / postsynapse / protein-containing complex assembly / Potential therapeutics for SARS / transmembrane transporter binding / membrane => GO:0016020 / endosome / protein domain specific binding / endoplasmic reticulum lumen / intracellular membrane-bounded organelle / glutamatergic synapse / lipid binding / synapse / protein-containing complex binding / protein kinase binding / Golgi apparatus / nucleoplasm / membrane
Similarity search - Function
AP-2 complex subunit sigma / Clathrin adaptor, alpha-adaptin, appendage, C-terminal subdomain / Adaptor protein complex AP-2, alpha subunit / Alpha adaptin AP2, C-terminal domain / Mu2, C-terminal domain / AP-2 complex subunit mu, N-terminal / Adaptor protein complex, sigma subunit / Clathrin adaptor, beta-adaptin, appendage, Ig-like subdomain / Beta-adaptin appendage, C-terminal subdomain / AP-1/2/4 complex subunit beta ...AP-2 complex subunit sigma / Clathrin adaptor, alpha-adaptin, appendage, C-terminal subdomain / Adaptor protein complex AP-2, alpha subunit / Alpha adaptin AP2, C-terminal domain / Mu2, C-terminal domain / AP-2 complex subunit mu, N-terminal / Adaptor protein complex, sigma subunit / Clathrin adaptor, beta-adaptin, appendage, Ig-like subdomain / Beta-adaptin appendage, C-terminal subdomain / AP-1/2/4 complex subunit beta / Beta2-adaptin appendage, C-terminal sub-domain / Beta2-adaptin appendage, C-terminal sub-domain / AP complex subunit beta / Clathrin adaptor complex, small chain / Clathrin adaptor complexes small chain signature. / Clathrin adaptor complexes medium chain signature 1. / Clathrin adaptor, mu subunit / Clathrin adaptor, mu subunit, conserved site / Clathrin adaptor complexes medium chain signature 2. / Coatomer/calthrin adaptor appendage, C-terminal subdomain / Adaptor complexes medium subunit family / AP complex, mu/sigma subunit / Clathrin adaptor complex small chain / AP-2 complex subunit mu, C-terminal superfamily / Mu homology domain / Mu homology domain (MHD) profile. / Clathrin adaptor, alpha/beta/gamma-adaptin, appendage, Ig-like subdomain / Adaptin C-terminal domain / Adaptin C-terminal domain / Clathrin/coatomer adaptor, adaptin-like, N-terminal / Adaptin N terminal region / Clathrin adaptor, appendage, Ig-like subdomain superfamily / Longin-like domain superfamily / TBP domain superfamily / Armadillo/beta-catenin-like repeats / Armadillo / Armadillo-like helical / Armadillo-type fold
Similarity search - Domain/homology
Trans-Golgi network integral membrane protein 2 / AP-2 complex subunit alpha-2 / AP-2 complex subunit sigma / AP-2 complex subunit beta / AP-2 complex subunit mu
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Methodsubtomogram averaging / cryo EM / Resolution: 10.2 Å
AuthorsKovtun O / Kane Dickson V / Kelly BT / Owen D / 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.
History
DepositionMar 12, 2020-
Header (metadata) releaseJul 29, 2020-
Map releaseJul 29, 2020-
UpdateApr 14, 2021-
Current statusApr 14, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.34
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.34
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10749.png

Downloads & links

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Map

FileDownload / File: emd_10749.map.gz / Format: CCP4 / Size: 2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map of AP2 dimer on membranes containing tyrosine-based cargo peptide
Voxel sizeX=Y=Z: 3.56 Å
Density
Contour LevelBy AUTHOR: 0.34 / Movie #1: 0.34
Minimum - Maximum-1.1544276 - 2.4761007
Average (Standard dev.)0.0024808054 (±0.15641883)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions808080
Spacing808080
CellA=B=C: 284.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.563.563.56
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z284.800284.800284.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS808080
D min/max/mean-1.1542.4760.002

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Supplemental data

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Mask #1

Fileemd_10749_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_10749_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_10749_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : AP2 dimer on membrane

EntireName: AP2 dimer on membrane
Components
  • Complex: AP2 dimer on membrane

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Supramolecule #1: AP2 dimer on membrane

SupramoleculeName: AP2 dimer on membrane / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Details: AP2 on membrane containing tyrosine-based cargo peptide
Source (natural)Organism: Rattus norvegicus (Norway rat)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation state3D array

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Sample preparation

Concentration0.3 mg/mL
BufferpH: 7.2
Component:
ConcentrationName
10.0 mMHepes KOH
120.0 mMPotassium Acetate
2.0 mMMagnesium chloride
5.0 mM2-Mercaptoethanol
GridModel: C-flat-2/2 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 98 % / Chamber temperature: 291 K / Instrument: LEICA EM GP
Details: The sample was supplemented with 10 nm nanogold fiducials, and 3 ul of the mixture was backside blotted for 3 seconds..
DetailsThe sample contained AP2 and 400 nm extruded liposomes

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 6.5 µm / Calibrated defocus min: 1.5 µm / Calibrated magnification: 81000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 6.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-10 / Number grids imaged: 2 / Average exposure time: 0.2 sec. / Average electron dose: 3.2 e/Å2
Details: The images were collected in movie mode at 10 frames per second
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 112237 / Number images used: 585669 / Reference model: reference-free / Method: geometrically defined initial positions / Software - Name: TOM
CTF correctionSoftware - Name: NOVACTF (ver. 1.0) / Details: CTF correction in novaCTF with by multiplication
Final angle assignmentType: OTHER / Software: (Name: TOM, AV3)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 10.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software: (Name: TOM, AV3) / Number subtomograms used: 22323
DetailsThe images were low pass filtered according to the cumulative radiation dose.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

2xa7

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: correlation coefficient

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