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- PDB-7oi5: Crystal structure of AP2 Mu2 - FCHO2 chimera (GST cleaved) -

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Basic information

Entry
Database: PDB / ID: 7oi5
TitleCrystal structure of AP2 Mu2 - FCHO2 chimera (GST cleaved)
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 / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / LDL clearance / Retrograde neurotrophin signalling / VLDLR internalisation and degradation / WNT5A-dependent internalization of FZD4 / extrinsic component of presynaptic endocytic zone membrane / MHC class II antigen presentation ...membrane invagination / Gap junction degradation / Formation of annular gap junctions / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / LDL clearance / Retrograde neurotrophin signalling / VLDLR internalisation and degradation / 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 / clathrin coat assembly / Recycling pathway of L1 / positive regulation of synaptic vesicle endocytosis / Cargo recognition for clathrin-mediated endocytosis / clathrin adaptor activity / Clathrin-mediated endocytosis / vesicle budding from membrane / clathrin-dependent endocytosis / signal sequence binding / 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 / negative regulation of protein localization to plasma membrane / 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 / synaptic vesicle / Cargo recognition for clathrin-mediated endocytosis / presynapse / Clathrin-mediated endocytosis / protein-containing complex assembly / cytoplasmic vesicle / transmembrane transporter binding / postsynapse / synapse / lipid binding / glutamatergic synapse / identical protein binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
F-BAR domain only protein 2 / : / GEM-interacting protein-like, FCH domain / Muniscin C-terminal / Muniscin C-terminal mu homology domain / Mu homology domain, subdomain B / Mu2, C-terminal domain / AP-2 complex subunit mu, N-terminal / Fes/CIP4 homology domain / FCH domain ...F-BAR domain only protein 2 / : / GEM-interacting protein-like, FCH domain / Muniscin C-terminal / Muniscin C-terminal mu homology domain / Mu homology domain, subdomain B / Mu2, C-terminal domain / AP-2 complex subunit mu, N-terminal / 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. / : / AP complex, mu/sigma subunit / Clathrin adaptor complex small chain / Adaptor complexes medium subunit family / AP-2 complex subunit mu, C-terminal superfamily / Mu homology domain / Mu homology domain (MHD) profile. / Longin-like domain superfamily / AH/BAR domain superfamily / Immunoglobulin-like / Sandwich / Mainly Beta
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: 2.61 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
D: AP-2 complex subunit mu,F-BAR domain only protein 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)79,0265
Polymers78,7502
Non-polymers2763
Water28816
1
B: AP-2 complex subunit mu,F-BAR domain only protein 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)39,5593
Polymers39,3751
Non-polymers1842
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
D: AP-2 complex subunit mu,F-BAR domain only protein 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)39,4672
Polymers39,3751
Non-polymers921
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)116.046, 55.380, 167.735
Angle α, β, γ (deg.)90.000, 109.050, 90.000
Int Tables number5
Space group name H-MC121
Space group name HallC2y
Symmetry operation#1: x,y,z
#2: -x,y,-z
#3: x+1/2,y+1/2,z
#4: -x+1/2,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: 39374.828 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) / Variant (production host): pLysS / References: UniProt: P84092, UniProt: Q0JRZ9
#2: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C3H8O3
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 16 / 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: 3.23 Å3/Da / Density % sol: 61.94 %
Crystal growTemperature: 289 K / Method: vapor diffusion, sitting drop / pH: 9.1
Details: 20% w/v PEG 3350, 0.2 M Sodium phosphate dibasic dehydrate pH 9.1. The crystal was cryo-protected by soaking in mother liquor supplemented with 25% glycerol.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I04-1 / Wavelength: 0.91589 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Jul 29, 2019
RadiationMonochromator: 0.91589 / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.91589 Å / Relative weight: 1
ReflectionResolution: 2.61→79.27 Å / Num. obs: 31089 / % possible obs: 99.9 % / Redundancy: 6.6 % / Biso Wilson estimate: 49.031 Å2 / CC1/2: 0.995 / Rmerge(I) obs: 0.163 / Rpim(I) all: 0.068 / Rrim(I) all: 0.177 / Net I/σ(I): 6.6
Reflection shellResolution: 2.61→2.65 Å / Rmerge(I) obs: 1.576 / Mean I/σ(I) obs: 1 / Num. unique obs: 1543 / CC1/2: 0.374 / Rpim(I) all: 0.693 / Rrim(I) all: 1.725 / % possible all: 99.3

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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: 2.61→79.27 Å / SU ML: 0.402 / Cross valid method: FREE R-VALUE / σ(F): 0.01 / Phase error: 37.3155
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2972 3014 5.04 %
Rwork0.2384 56774 -
obs0.2414 29463 99.49 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 73.33 Å2
Refinement stepCycle: LAST / Resolution: 2.61→79.27 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4635 0 18 16 4669
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00394746
X-RAY DIFFRACTIONf_angle_d0.72776373
X-RAY DIFFRACTIONf_chiral_restr0.0507694
X-RAY DIFFRACTIONf_plane_restr0.0096801
X-RAY DIFFRACTIONf_dihedral_angle_d6.9433629
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.61-2.650.39721150.39492589X-RAY DIFFRACTION98.87
2.65-2.690.42131620.37092570X-RAY DIFFRACTION99.24
2.69-2.740.43491580.3672555X-RAY DIFFRACTION98.73
2.74-2.790.45831580.37122558X-RAY DIFFRACTION99.05
2.79-2.840.4666990.35492597X-RAY DIFFRACTION99.7
2.84-2.90.39481290.3512634X-RAY DIFFRACTION99.5
2.9-2.960.39781310.32862508X-RAY DIFFRACTION99.66
2.97-3.030.43861570.31092629X-RAY DIFFRACTION99.61
3.03-3.110.31961290.28632535X-RAY DIFFRACTION99.48
3.11-3.190.38141070.27862610X-RAY DIFFRACTION98.91
3.19-3.290.34211550.29542551X-RAY DIFFRACTION99.34
3.29-3.390.31771470.27592606X-RAY DIFFRACTION99.57
3.39-3.520.29361650.23782529X-RAY DIFFRACTION99.67
3.52-3.660.29561170.22022606X-RAY DIFFRACTION99.6
3.66-3.820.29821220.22482582X-RAY DIFFRACTION99.82
3.82-4.020.31061280.22452655X-RAY DIFFRACTION99.82
4.02-4.280.26751450.19532573X-RAY DIFFRACTION99.82
4.28-4.610.25341420.1812606X-RAY DIFFRACTION99.78
4.61-5.070.21861340.15822537X-RAY DIFFRACTION99.85
5.07-5.80.25551500.18812588X-RAY DIFFRACTION99.74
5.8-7.310.27841440.21872559X-RAY DIFFRACTION99.82
7.31-79.270.21081200.21912597X-RAY DIFFRACTION99.31

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