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- PDB-7ohz: Crystal structure of AP2 Mu2 - FCHO2 chimera (His6-tagged) -

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Basic information

Entry
Database: PDB / ID: 7ohz
TitleCrystal structure of AP2 Mu2 - FCHO2 chimera (His6-tagged)
ComponentsAP-2 complex subunit mu,F-BAR domain only protein 2
KeywordsENDOCYTOSIS / clathrin-mediated endocytosis (CME) / protein recycling / plasma membrane
Function / homology
Function and homology information


membrane invagination / Gap junction degradation / Formation of annular gap junctions / presynaptic endocytic zone membrane / 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 ...membrane invagination / Gap junction degradation / Formation of annular gap junctions / presynaptic endocytic zone membrane / 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 / clathrin coat assembly / 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 / clathrin-coated vesicle / low-density lipoprotein particle receptor binding / phosphatidylserine binding / Trafficking of GluR2-containing AMPA receptors / positive regulation of receptor internalization / synaptic vesicle endocytosis / clathrin-coated pit / phosphatidylinositol-4,5-bisphosphate binding / phosphatidylinositol binding / protein localization to plasma membrane / intracellular protein transport / terminal bouton / receptor internalization / disordered domain specific binding / Cargo recognition for clathrin-mediated endocytosis / Clathrin-mediated endocytosis / cytoplasmic vesicle / postsynapse / protein-containing complex assembly / transmembrane transporter binding / glutamatergic synapse / lipid binding / synapse / identical protein binding / plasma membrane / cytoplasm / cytosol
Similarity search - Function
F-BAR domain only protein 2 / Muniscin C-terminal / Muniscin C-terminal mu homology domain / Mu2, C-terminal domain / AP-2 complex subunit mu, N-terminal / Fes/CIP4, and EFC/F-BAR homology domain / Fes/CIP4 homology domain / FCH domain / F-BAR domain / F-BAR domain profile. ...F-BAR domain only protein 2 / Muniscin C-terminal / Muniscin C-terminal mu homology domain / Mu2, C-terminal domain / AP-2 complex subunit mu, N-terminal / Fes/CIP4, and EFC/F-BAR homology domain / Fes/CIP4 homology domain / FCH domain / F-BAR domain / F-BAR domain profile. / 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 / AH/BAR domain superfamily
Similarity search - Domain/homology
AP-2 complex subunit mu / F-BAR domain only protein 2
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Homo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.27 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
Citation
Journal: 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.
#1: Journal: Acta Crystallogr., Sect. D: Biol. Crystallogr. / Year: 2012
Title: Towards automated crystallographic structure refinement with phenix.refine.
Authors: Afonine, P.V.
History
DepositionMay 11, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 1, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: AP-2 complex subunit mu,F-BAR domain only protein 2
A: AP-2 complex subunit mu,F-BAR domain only protein 2


Theoretical massNumber of molelcules
Total (without water)79,5962
Polymers79,5962
Non-polymers00
Water2,612145
1
B: AP-2 complex subunit mu,F-BAR domain only protein 2


Theoretical massNumber of molelcules
Total (without water)39,7981
Polymers39,7981
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
A: AP-2 complex subunit mu,F-BAR domain only protein 2


Theoretical massNumber of molelcules
Total (without water)39,7981
Polymers39,7981
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)55.683, 129.861, 64.423
Angle α, β, γ (deg.)90.000, 102.475, 90.000
Int Tables number4
Space group name H-MP1211
Space group name HallP2yb
Symmetry operation#1: x,y,z
#2: -x,y+1/2,-z

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Components

#1: Protein AP-2 complex subunit mu,F-BAR domain only protein 2 / 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: 39798.238 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat), (gene. exp.) Homo sapiens (human)
Gene: Ap2m1, FCHO2 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21 (pLysS) / References: UniProt: P84092, UniProt: Q0JRZ9
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 145 / Source method: isolated from a natural source / Formula: H2O

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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: 2.85 Å3/Da / Density % sol: 56.91 %
Crystal growTemperature: 289 K / Method: vapor diffusion, sitting drop / pH: 7
Details: 20% w/v PEG 3,350 0.2 M DL-Malic acid pH 7.0. The crystals were cryo-protected by soaking in mother liquor supplemented with 30-32% glycerol.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I04 / Wavelength: 0.97953 Å
DetectorType: DECTRIS EIGER2 XE 16M / Detector: PIXEL / Date: Jul 1, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97953 Å / Relative weight: 1
ReflectionResolution: 2.27→62.9 Å / Num. obs: 40099 / % possible obs: 97.1 % / Redundancy: 6.3 % / Biso Wilson estimate: 38.15 Å2 / CC1/2: 0.996 / Rmerge(I) obs: 0.136 / Rpim(I) all: 0.058 / Rrim(I) all: 0.148 / Net I/σ(I): 7
Reflection shellResolution: 2.27→2.31 Å / Redundancy: 4.5 % / Rmerge(I) obs: 1.165 / Mean I/σ(I) obs: 1.2 / Num. unique obs: 1520 / Rpim(I) all: 0.593 / Rrim(I) all: 1.318 / % possible all: 72.4

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Processing

Software
NameVersionClassification
REFMAC5.8.0257refinement
PHENIX1.19rc1_4016refinement
DIALSdata reduction
DIALSdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: Phaser

Resolution: 2.27→62.9 Å / SU ML: 0.4086 / Cross valid method: FREE R-VALUE / σ(F): 0.14 / Phase error: 37.769
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.3139 3864 4.94 %
Rwork0.2672 74342 -
obs0.2695 38073 95.93 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 53.56 Å2
Refinement stepCycle: LAST / Resolution: 2.27→62.9 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4616 0 0 147 4763
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0034720
X-RAY DIFFRACTIONf_angle_d0.6436348
X-RAY DIFFRACTIONf_chiral_restr0.0487685
X-RAY DIFFRACTIONf_plane_restr0.0049803
X-RAY DIFFRACTIONf_dihedral_angle_d5.8205620
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.27-2.30.3961130.38971872X-RAY DIFFRACTION66.61
2.3-2.330.37771100.37592017X-RAY DIFFRACTION73.65
2.33-2.360.42541050.37082288X-RAY DIFFRACTION82.09
2.36-2.390.42741220.37112555X-RAY DIFFRACTION91.93
2.39-2.420.38251590.36222656X-RAY DIFFRACTION95.78
2.42-2.460.42351050.33782709X-RAY DIFFRACTION97.17
2.46-2.50.38771600.32962632X-RAY DIFFRACTION97.72
2.5-2.540.38661620.34222741X-RAY DIFFRACTION97.71
2.54-2.580.37621470.32322663X-RAY DIFFRACTION97.4
2.58-2.630.32571440.31242710X-RAY DIFFRACTION97.77
2.63-2.680.36451370.30652736X-RAY DIFFRACTION98.19
2.68-2.740.4121370.30582686X-RAY DIFFRACTION98.16
2.74-2.790.36091260.31532787X-RAY DIFFRACTION99.28
2.79-2.860.35521290.28552733X-RAY DIFFRACTION99
2.86-2.930.35331460.26822775X-RAY DIFFRACTION99.66
2.93-3.010.36031500.28332708X-RAY DIFFRACTION99.34
3.01-3.10.27781320.26692767X-RAY DIFFRACTION99.55
3.1-3.20.36011580.26042726X-RAY DIFFRACTION99.24
3.2-3.310.35871140.27382790X-RAY DIFFRACTION99.66
3.31-3.450.26041240.26332783X-RAY DIFFRACTION99.76
3.45-3.60.30021450.24552763X-RAY DIFFRACTION99.66
3.6-3.790.31741180.22992791X-RAY DIFFRACTION99.76
3.79-4.030.29341420.24322758X-RAY DIFFRACTION99.93
4.03-4.340.29861430.22982745X-RAY DIFFRACTION99.79
4.34-4.780.21211720.21822713X-RAY DIFFRACTION99.76
4.78-5.470.29691440.23342778X-RAY DIFFRACTION99.9
5.47-6.890.32351420.25382770X-RAY DIFFRACTION99.83
6.89-62.90.2841780.27052690X-RAY DIFFRACTION98.59

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