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- PDB-7z5c: Chimera of AP2 with FCHO2 linker domain as a fusion on Cmu2 subunit -

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Basic information

Entry
Database: PDB / ID: 7z5c
TitleChimera of AP2 with FCHO2 linker domain as a fusion on Cmu2 subunit
Components
  • AP-2 complex subunit alpha-2
  • AP-2 complex subunit beta
  • AP-2 complex subunit mu
  • AP-2 complex subunit sigma
KeywordsPROTEIN TRANSPORT / adaptor complex / AP2 / trafficking / clathrin / CCP
Function / homology
Function and homology information


Gap junction degradation / Formation of annular gap junctions / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / LDL clearance / 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 ...Gap junction degradation / Formation of annular gap junctions / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / LDL clearance / 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 / 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 / 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 / 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 / vesicle-mediated transport / MHC class II antigen presentation / VLDLR internalisation and degradation / phosphatidylinositol binding / kidney development / clathrin-coated endocytic vesicle membrane / intracellular protein transport / terminal bouton / cytoplasmic side of plasma membrane / receptor internalization / kinase binding / 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 / protein domain specific binding / intracellular membrane-bounded organelle / lipid binding / glutamatergic synapse / synapse / protein-containing complex binding / protein kinase binding / membrane / plasma membrane / cytosol
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
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)
Homo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.16 Å
AuthorsKane Dickson, V. / Qu, K. / Owen, D.J. / Briggs, J.A. / Zaccai, N.R.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust United Kingdom
CitationJournal: Sci Adv / Year: 2022
Title: FCHO controls AP2's initiating role in endocytosis through a PtdIns(4,5)P-dependent switch.
Authors: Nathan R Zaccai / Zuzana Kadlecova / Veronica Kane Dickson / Kseniya Korobchevskaya / Jan Kamenicky / Oleksiy Kovtun / Perunthottathu K Umasankar / Antoni G Wrobel / Jonathan G G Kaufman / ...Authors: Nathan R Zaccai / Zuzana Kadlecova / Veronica Kane Dickson / Kseniya Korobchevskaya / Jan Kamenicky / Oleksiy Kovtun / Perunthottathu K Umasankar / Antoni G Wrobel / Jonathan G G Kaufman / Sally R Gray / Kun Qu / Philip R Evans / Marco Fritzsche / Filip Sroubek / Stefan Höning / John A G Briggs / Bernard T Kelly / David J Owen / Linton M Traub /
Abstract: Clathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo adaptor AP2 requires membrane- ...Clathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo adaptor AP2 requires membrane-localized Fer/Cip4 homology domain-only proteins (FCHO). Here, live-cell enhanced total internal reflection fluorescence-structured illumination microscopy shows that FCHO marks sites of clathrin-coated pit (CCP) initiation, which mature into uniform-sized CCPs comprising a central patch of AP2 and clathrin corralled by an FCHO/Epidermal growth factor potential receptor substrate number 15 (Eps15) ring. We dissect the network of interactions between the FCHO interdomain linker and AP2, which concentrates, orients, tethers, and partially destabilizes closed AP2 at the plasma membrane. AP2's subsequent membrane deposition drives its opening, which triggers FCHO displacement through steric competition with phosphatidylinositol 4,5-bisphosphate, clathrin, cargo, and CME accessory factors. FCHO can now relocate toward a CCP's outer edge to engage and activate further AP2s to drive CCP growth/maturation.
History
DepositionMar 9, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 11, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: AP-2 complex subunit alpha-2
B: AP-2 complex subunit beta
M: AP-2 complex subunit mu
S: AP-2 complex subunit sigma


Theoretical massNumber of molelcules
Total (without water)203,3754
Polymers203,3754
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area19470 Å2
ΔGint-73 kcal/mol
Surface area70430 Å2

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Components

#1: Protein AP-2 complex subunit alpha-2 / 100 kDa coated vesicle protein C / Adaptor protein complex AP-2 subunit alpha-2 / Adaptor-related ...100 kDa coated vesicle protein C / Adaptor protein complex AP-2 subunit alpha-2 / Adaptor-related protein complex 2 subunit alpha-2 / Alpha-adaptin C / Alpha2-adaptin / Clathrin assembly protein complex 2 alpha-C large chain / Plasma membrane adaptor HA2/AP2 adaptin alpha C subunit


Mass: 69656.297 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Ap2a2, Adtab
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P18484
#2: Protein AP-2 complex subunit beta / AP105B / Adaptor protein complex AP-2 subunit beta / Adaptor-related protein complex 2 subunit beta ...AP105B / Adaptor protein complex AP-2 subunit beta / Adaptor-related protein complex 2 subunit beta / Beta-2-adaptin / Beta-adaptin / Clathrin assembly protein complex 2 beta large chain / Plasma membrane adaptor HA2/AP2 adaptin beta subunit


Mass: 66953.195 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: AP2B1, ADTB2, CLAPB1
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P63010
#3: Protein AP-2 complex subunit mu / AP-2 mu chain / Adaptor protein complex AP-2 subunit mu / Adaptor-related protein complex 2 subunit ...AP-2 mu chain / Adaptor protein complex AP-2 subunit mu / Adaptor-related protein complex 2 subunit mu / Clathrin assembly protein complex 2 mu medium chain / Clathrin coat assembly protein AP50 / Clathrin coat-associated protein AP50 / Mu2-adaptin / Plasma membrane adaptor AP-2 50 kDa protein


Mass: 49726.641 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Ap2m1
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P84092
#4: Protein AP-2 complex subunit sigma / Adaptor protein complex AP-2 subunit sigma / Adaptor-related protein complex 2 subunit sigma / ...Adaptor protein complex AP-2 subunit sigma / Adaptor-related protein complex 2 subunit sigma / Clathrin assembly protein 2 sigma small chain / Clathrin coat assembly protein AP17 / Clathrin coat-associated protein AP17 / Plasma membrane adaptor AP-2 17 kDa protein / Sigma-adaptin 3b / Sigma2-adaptin


Mass: 17038.688 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ap2s1, Ap17, Claps2
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P62743

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: AP2 core complex / Type: COMPLEX
Details: Ternary complex of AP2 core expressed as part of a chimaera with FCHO2 linker (not modelled)
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.203 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 7.6
Details: 50% HKT buffer (10mM Hepes, 10mM Tris 120mM potassium acetate pH 7.2) and 50% Core buffer (10mM Tris, 250mM NaCl, pH 8)
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2900 nm / Nominal defocus min: 900 nm
Image recordingElectron dose: 47 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.16 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 143815 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00213951
ELECTRON MICROSCOPYf_angle_d0.518886
ELECTRON MICROSCOPYf_dihedral_angle_d3.9821868
ELECTRON MICROSCOPYf_chiral_restr0.042198
ELECTRON MICROSCOPYf_plane_restr0.0032403

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