EMDB-10750: AP2 in clathrin coats assembled on a membrane containing tyrosine...

Basic information

• Entry: Database: EMDB / ID: EMD-10750
• Title: AP2 in clathrin coats assembled on a membrane containing tyrosine-based cargo peptide
• Map data: Sharpened map of AP2 in clathrin coat formed on membranes containing tyrosine-based cargo peptides

Sample

• Complex: Clathrin/AP2 coat assembled on AP2 membrane.
  • Complex: Clathrin
  • Complex: AP2

Function / homology

Function:

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 / Retrograde neurotrophin signalling / VLDLR internalisation and degradation / 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 FZD4 / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / Trafficking of GluR2-containing AMPA receptors / extrinsic component of presynaptic endocytic zone membrane / VLDLR internalisation and degradation / clathrin adaptor complex / WNT5A-dependent internalization of FZD4 / 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 / Cargo recognition for clathrin-mediated endocytosis / Retrograde neurotrophin signalling / positive regulation of synaptic vesicle endocytosis / clathrin-coated endocytic vesicle / Clathrin-mediated endocytosis / Cargo recognition for clathrin-mediated endocytosis / clathrin adaptor activity / Clathrin-mediated endocytosis / LDL clearance / vesicle budding from membrane / membrane coat / clathrin-dependent endocytosis / trans-Golgi network transport vesicle / MHC class II antigen presentation / coronary vasculature development / signal sequence binding / Nef Mediated CD4 Down-regulation / endolysosome membrane / aorta development / ventricular septum development / Neutrophil degranulation / clathrin binding / low-density lipoprotein particle receptor binding / Golgi Associated Vesicle Biogenesis / Trafficking of GluR2-containing AMPA receptors / Recycling pathway of L1 / positive regulation of receptor internalization / EPH-ephrin mediated repulsion of cells / synaptic vesicle endocytosis / negative regulation of protein localization to plasma membrane / protein serine/threonine kinase binding / clathrin-coated pit / transport vesicle / vesicle-mediated transport / MHC class II antigen presentation / phosphatidylinositol binding / VLDLR internalisation and degradation / kidney development / Post-translational protein phosphorylation / intracellular protein transport / trans-Golgi network / clathrin-coated endocytic vesicle membrane / terminal bouton / receptor internalization / kinase binding / cytoplasmic side of plasma membrane / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / disordered domain specific binding / endocytic vesicle membrane / Cargo recognition for clathrin-mediated endocytosis / synaptic vesicle / presynapse / Clathrin-mediated endocytosis / protein-containing complex assembly / cytoplasmic vesicle / transmembrane transporter binding / Potential therapeutics for SARS / postsynapse / endosome / protein domain specific binding / endoplasmic reticulum lumen / lipid binding / synapse / protein-containing complex binding / protein kinase binding / glutamatergic synapse / Golgi apparatus / nucleoplasm / membrane / plasma membrane / cytosol

Domain/homology:

Keratinocyte-associated gene product / 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 / Clathrin adaptor, beta-adaptin, appendage, Ig-like subdomain / Beta2-adaptin appendage, C-terminal sub-domain / AP-1/2/4 complex subunit beta / Adaptor protein complex, sigma subunit / : / Beta-adaptin appendage, C-terminal subdomain / 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 / Clathrin adaptor, alpha/beta/gamma-adaptin, appendage, Ig-like subdomain / Adaptin C-terminal domain / Adaptin C-terminal domain / 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/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

Component:

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

• Biological species: Sus scrofa (pig) / Homo sapiens (human)
• Method: subtomogram averaging / cryo EM / Resolution: 11.9 Å
• Authors: Kovtun O / Kane Dickson V / Kelly BT / Owen D / Briggs JAG

Funding support

United Kingdom, 2 items

Organization	Grant number	Country
Wellcome Trust	207455/Z/17/Z	United Kingdom
Medical Research Council (MRC, United Kingdom)	MC_UP_1201/16	United Kingdom

• Citation: Journal: 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 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

Deposition	Mar 12, 2020	-
Header (metadata) release	Jul 29, 2020	-
Map release	Jul 29, 2020	-
Update	Apr 14, 2021	-
Current status	Apr 14, 2021	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_10750.map.gz / Format: CCP4 / Size: 6.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Sharpened map of AP2 in clathrin coat formed on membranes containing tyrosine-based cargo peptides
• Voxel size: X=Y=Z: 1.78 Å

Density

Contour Level	By AUTHOR: 0.32 / Movie #1: 0.32
Minimum - Maximum	-2.501963 - 2.8607073
Average (Standard dev.)	0.0015377516 (±0.22353551)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 120 120 120 Spacing 120 120 120
Cell	A=B=C: 213.59999 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	1.78	1.78	1.78
M x/y/z	120	120	120
origin x/y/z	0.000	0.000	0.000
length x/y/z	213.600	213.600	213.600
α/β/γ	90.000	90.000	90.000
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	120	120	120
D min/max/mean	-2.502	2.861	0.002

Supplemental data

Mask #1

• File: emd_10750_msk_1.map

Half map: #1

• File: emd_10750_half_map_1.map

Half map: #2

• File: emd_10750_half_map_2.map

Sample components

Entire : Clathrin/AP2 coat assembled on AP2 membrane.

• Entire: Name: Clathrin/AP2 coat assembled on AP2 membrane.

Components

• Complex: Clathrin/AP2 coat assembled on AP2 membrane.
  • Complex: Clathrin
  • Complex: AP2

Supramolecule #1: Clathrin/AP2 coat assembled on AP2 membrane.

• Supramolecule: Name: Clathrin/AP2 coat assembled on AP2 membrane. / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4; Details: Clathrin/AP coat was formed on the membrane containing dileucine- and tyrosine-based cargo signal peptides. The AP2 adaptor lacked hinge and appendage regions in its alpha subunit.

Supramolecule #2: Clathrin

• Supramolecule: Name: Clathrin / type: complex / ID: 2 / Parent: 1 ; Details: Clathrin in the clathrin/AP2 coat formed on the membrane
• Source (natural): Organism: Sus scrofa (pig) / Organ: brain

Supramolecule #3: AP2

• Supramolecule: Name: AP2 / type: complex / ID: 3 / Parent: 1 ; Details: AP2 in clathrin coat formed on membranes containing dileucine- and tyrosine-based cargo peptides
• Source (natural): Organism: Homo sapiens (human)
• Recombinant expression: Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

Experimental details

Structure determination

• Method: cryo EM
• Processing: subtomogram averaging
• Aggregation state: 3D array

Sample preparation

• Concentration: 0.7 mg/mL

Buffer

pH: 7.2
Component:

Concentration	Name
10.0 mM	Hepes KOH
120.0 mM	Potassium Acetate
2.0 mM	Magnesium chloride
5.0 mM	2-Mercaptoethanol

• Grid: Model: C-flat-2/2 / Support film - Material: CARBON / Support film - topology: HOLEY
• Vitrification: Cryogen 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..
• Details: The sample (in vitro budding reaction) contained AP2, clathrin and 400 nm extruded liposomes

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Specialist optics: Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
• Image recording: Film 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/Ų; Details: The images were collected in movie mode at 10 frames per second
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: C2 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 FIELD / Cs: 2.7 mm / Nominal defocus max: 6.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 81000
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Details: The images were low pass filtered according to the cumulative radiation dose.
• Final reconstruction: Applied symmetry - Point group: C₁ (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 11.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software: (Name: TOM, AV3) / Number subtomograms used: 14805
• Extraction: Number tomograms: 25694 / Number images used: 224577 / Reference model: reference-free; Method: geometrically defined initial positions on membrane strctures; Software - Name: TOM
• CTF correction: Software - Name: NOVACTF (ver. 1.0) / Details: CTF correction in novaCTF with by multiplication
• Final angle assignment: Type: OTHER / Software: (Name: TOM, AV3)

Atomic model buiding 1

Initial model

PDB ID:

1xa7

• Refinement: Space: REAL / Protocol: FLEXIBLE FIT / Target criteria: correlation coefficient