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- PDB-7ofp: Apo Structure of Mu2 Adaptin Subunit (Ap50) Of AP2 Clathrin Adaptor -

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Basic information

Entry
Database: PDB / ID: 7ofp
TitleApo Structure of Mu2 Adaptin Subunit (Ap50) Of AP2 Clathrin Adaptor
ComponentsAP-2 complex subunit mu
KeywordsENDOCYTOSIS / clathrin-mediated endocytosis (CME) / YxxPhi motif / Pi(4 / 5)P2 / protein recycling / plasma membrane
Function / homology
Function and homology information


Gap junction degradation / Formation of annular gap junctions / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / LDL clearance / VLDLR internalisation and degradation / Retrograde neurotrophin signalling / WNT5A-dependent internalization of FZD4 / extrinsic component of presynaptic endocytic zone membrane / MHC class II antigen presentation / AP-2 adaptor complex ...Gap junction degradation / Formation of annular gap junctions / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / LDL clearance / VLDLR internalisation and degradation / Retrograde neurotrophin signalling / WNT5A-dependent internalization of FZD4 / extrinsic component of presynaptic endocytic zone membrane / MHC class II antigen presentation / AP-2 adaptor complex / regulation of vesicle size / postsynaptic neurotransmitter receptor internalization / Recycling pathway of L1 / Cargo recognition for clathrin-mediated endocytosis / positive regulation of synaptic vesicle endocytosis / Clathrin-mediated endocytosis / clathrin adaptor activity / vesicle budding from membrane / clathrin-dependent endocytosis / signal sequence binding / negative regulation of protein localization to plasma membrane / low-density lipoprotein particle receptor binding / Trafficking of GluR2-containing AMPA receptors / positive regulation of receptor internalization / synaptic vesicle endocytosis / clathrin-coated pit / intracellular protein transport / terminal bouton / receptor internalization / disordered domain specific binding / cytoplasmic vesicle / postsynapse / protein-containing complex assembly / transmembrane transporter binding / synapse / glutamatergic synapse / lipid binding / plasma membrane
Similarity search - Function
Mu2, C-terminal domain / AP-2 complex subunit mu, N-terminal / Clathrin adaptor complexes medium chain signature 1. / Clathrin adaptor, mu subunit / Clathrin adaptor, mu subunit, conserved site / Clathrin adaptor complexes medium chain signature 2. / Adaptor complexes medium subunit family / AP complex, mu/sigma subunit / Clathrin adaptor complex small chain / AP-2 complex subunit mu, C-terminal superfamily ...Mu2, C-terminal domain / AP-2 complex subunit mu, N-terminal / Clathrin adaptor complexes medium chain signature 1. / Clathrin adaptor, mu subunit / Clathrin adaptor, mu subunit, conserved site / Clathrin adaptor complexes medium chain signature 2. / 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. / Longin-like domain superfamily
Similarity search - Domain/homology
CITRIC ACID / AP-2 complex subunit mu
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.92 Å
AuthorsZaccai, N.R. / Kelly, B.T. / Evans, P.R. / Owen, D.J.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust207455/Z/17/Z 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
DepositionMay 5, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 1, 2022Provider: repository / Type: Initial release
Revision 1.1Jan 31, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: AP-2 complex subunit mu
B: AP-2 complex subunit mu
hetero molecules


Theoretical massNumber of molelcules
Total (without water)66,53812
Polymers65,5172
Non-polymers1,02110
Water4,900272
1
A: AP-2 complex subunit mu
hetero molecules


Theoretical massNumber of molelcules
Total (without water)33,5038
Polymers32,7581
Non-polymers7457
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: AP-2 complex subunit mu
hetero molecules


Theoretical massNumber of molelcules
Total (without water)33,0354
Polymers32,7581
Non-polymers2763
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)123.830, 123.830, 112.668
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number170
Space group name H-MP65
Space group name HallP65
Symmetry operation#1: x,y,z
#2: x-y,x,z+5/6
#3: y,-x+y,z+1/6
#4: -y,x-y,z+2/3
#5: -x+y,-x,z+1/3
#6: -x,-y,z+1/2

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Components

#1: 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: 32758.363 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Ap2m1 / Plasmid: pmW / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21 (plysS) / References: UniProt: P84092
#2: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: C3H8O3
#3: Chemical ChemComp-CIT / CITRIC ACID / Citric acid


Mass: 192.124 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H8O7
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 272 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.81 Å3/Da / Density % sol: 67.68 %
Crystal growTemperature: 289 K / Method: vapor diffusion, hanging drop / pH: 7
Details: Protein (10 mg/ml) and peptide (DVDEEGYSIKPETNQNDTKENHFYSS) (2mg/ml) were equilibrated against 1.5M Ammonium Sulphate 0.1M Hepes pH 7.0. Crystals were cryo-protected by soaking in mother ...Details: Protein (10 mg/ml) and peptide (DVDEEGYSIKPETNQNDTKENHFYSS) (2mg/ml) were equilibrated against 1.5M Ammonium Sulphate 0.1M Hepes pH 7.0. Crystals were cryo-protected by soaking in mother liquor supplemented with 20% glycerol and peptide (peptide did not crystallise).
Temp details: Room Temperature

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I04-1 / Wavelength: 0.91587 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Apr 13, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.91587 Å / Relative weight: 1
ReflectionResolution: 1.92→77.68 Å / Num. obs: 74711 / % possible obs: 99.89 % / Redundancy: 17.7 % / Biso Wilson estimate: 30.59 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.223 / Rpim(I) all: 0.055 / Rrim(I) all: 0.23 / Net I/σ(I): 13.1
Reflection shellResolution: 1.92→1.95 Å / Redundancy: 12.1 % / Rmerge(I) obs: 6.379 / Mean I/σ(I) obs: 1 / Num. unique obs: 3642 / CC1/2: 0.44 / Rpim(I) all: 1.912 / Rrim(I) all: 6.664 / % possible all: 98

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Processing

Software
NameVersionClassification
REFMAC1.19rc1_4016refinement
PHENIX1.19rc1_4016refinement
DIALSdata reduction
DIALSdata scaling
PHASERphasing
STARANISOdata processing
autoPROCdata processing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1BXX

1bxx


Resolution: 1.92→77.68 Å / SU ML: 0.2223 / Cross valid method: FREE R-VALUE / σ(F): 0.21 / Phase error: 23.7951
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2105 7527 5.11 %
Rwork0.188 139851 -
obs0.1892 74655 99.83 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 41.41 Å2
Refinement stepCycle: LAST / Resolution: 1.92→77.68 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4252 0 67 272 4591
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01834404
X-RAY DIFFRACTIONf_angle_d1.25415910
X-RAY DIFFRACTIONf_chiral_restr0.0854647
X-RAY DIFFRACTIONf_plane_restr0.0116738
X-RAY DIFFRACTIONf_dihedral_angle_d9.8311605
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.92-1.940.35042300.32634572X-RAY DIFFRACTION96.83
1.94-1.960.34843020.31664572X-RAY DIFFRACTION99.9
1.96-1.990.31872460.30294669X-RAY DIFFRACTION99.31
1.99-2.010.31532360.28714687X-RAY DIFFRACTION99.96
2.01-2.040.28132460.27584680X-RAY DIFFRACTION99.9
2.04-2.070.30682620.26914635X-RAY DIFFRACTION99.98
2.07-2.10.28652820.25774639X-RAY DIFFRACTION99.94
2.1-2.130.28423060.25024609X-RAY DIFFRACTION100
2.13-2.160.26512300.24834708X-RAY DIFFRACTION99.96
2.16-2.20.24972810.22994590X-RAY DIFFRACTION99.96
2.2-2.240.29312160.21724734X-RAY DIFFRACTION99.88
2.24-2.280.23412700.20684620X-RAY DIFFRACTION99.96
2.28-2.320.24292450.20224653X-RAY DIFFRACTION99.96
2.32-2.370.22611960.20754750X-RAY DIFFRACTION99.9
2.37-2.420.20073040.19394597X-RAY DIFFRACTION99.98
2.42-2.480.20142760.19834662X-RAY DIFFRACTION99.98
2.48-2.540.23052380.1854684X-RAY DIFFRACTION100
2.54-2.610.21521680.19124744X-RAY DIFFRACTION100
2.61-2.680.22062860.18874652X-RAY DIFFRACTION100
2.68-2.770.22462180.20144684X-RAY DIFFRACTION100
2.77-2.870.20452400.18154680X-RAY DIFFRACTION100
2.87-2.980.21212780.18214652X-RAY DIFFRACTION99.98
2.98-3.120.22512580.17844646X-RAY DIFFRACTION100
3.12-3.280.19582400.16894696X-RAY DIFFRACTION99.98
3.28-3.490.18212410.17074694X-RAY DIFFRACTION100
3.49-3.760.20241920.15654721X-RAY DIFFRACTION100
3.76-4.140.17652700.14974661X-RAY DIFFRACTION100
4.14-4.730.15912960.13734600X-RAY DIFFRACTION99.92
4.73-5.960.16542370.15984703X-RAY DIFFRACTION100
5.97-77.680.22052370.21974657X-RAY DIFFRACTION99.84

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