+Open data
-Basic information
Entry | Database: PDB / ID: 6owo | ||||||
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Title | CRYO-EM STRUCTURE OF PHOSPHORYLATED AP-2 CORE BOUND TO NECAP | ||||||
Components |
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Keywords | ENDOCYTOSIS / AP2 / NECAP2 PROTEIN / CLATHRIN VESICLE / LIPID-BINDING / ADAPTOR / MEMBRANE / TRANSPORT / PHOSPHORYLATION | ||||||
Function / homology | Function and homology information Formation of annular gap junctions / Gap junction degradation / clathrin vesicle coat / 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 / VLDLR internalisation and degradation / Retrograde neurotrophin signalling ...Formation of annular gap junctions / Gap junction degradation / clathrin vesicle coat / 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 / VLDLR internalisation and degradation / Retrograde neurotrophin signalling / Trafficking of GluR2-containing AMPA receptors / Retrograde neurotrophin signalling / clathrin adaptor complex / WNT5A-dependent internalization of FZD4 / clathrin coat / extrinsic component of presynaptic endocytic zone membrane / VLDLR internalisation and degradation / cardiac septum development / 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 / clathrin coat assembly / Cargo recognition for clathrin-mediated endocytosis / positive regulation of synaptic vesicle endocytosis / Clathrin-mediated endocytosis / clathrin adaptor activity / Clathrin-mediated endocytosis / vesicle budding from membrane / membrane coat / clathrin-dependent endocytosis / MHC class II antigen presentation / coronary vasculature development / neurotransmitter receptor internalization / positive regulation of protein localization to membrane / signal sequence binding / aorta development / negative regulation of protein localization to plasma membrane / regulation of hematopoietic stem cell differentiation / ventricular septum development / low-density lipoprotein particle receptor binding / clathrin binding / Trafficking of GluR2-containing AMPA receptors / positive regulation of endocytosis / positive regulation of receptor internalization / synaptic vesicle endocytosis / protein serine/threonine kinase binding / vesicle-mediated transport / Neutrophil degranulation / phosphatidylinositol binding / secretory granule / kidney development / intracellular protein transport / cytoplasmic side of plasma membrane / kinase binding / endocytosis / disordered domain specific binding / protein transport / synaptic vesicle / heart development / cytoplasmic vesicle / postsynapse / protein-containing complex assembly / transmembrane transporter binding / protein domain specific binding / intracellular membrane-bounded organelle / glutamatergic synapse / lipid binding / synapse / protein-containing complex binding / protein kinase binding / mitochondrion / plasma membrane Similarity search - Function | ||||||
Biological species | Mus musculus (house mouse) Rattus norvegicus (Norway rat) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å | ||||||
Authors | Partlow, E.A. / Baker, R.W. / Beacham, G.M. / Chappie, J.S. / Leschziner, A.E. / Hollopeter, G. | ||||||
Funding support | United States, 1items
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Citation | Journal: Elife / Year: 2019 Title: A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex. Authors: Edward A Partlow / Richard W Baker / Gwendolyn M Beacham / Joshua S Chappie / Andres E Leschziner / Gunther Hollopeter / Abstract: Endocytosis of transmembrane proteins is orchestrated by the AP2 clathrin adaptor complex. AP2 dwells in a closed, inactive state in the cytosol, but adopts an open, active conformation on the plasma ...Endocytosis of transmembrane proteins is orchestrated by the AP2 clathrin adaptor complex. AP2 dwells in a closed, inactive state in the cytosol, but adopts an open, active conformation on the plasma membrane. Membrane-activated complexes are also phosphorylated, but the significance of this mark is debated. We recently proposed that NECAP negatively regulates AP2 by binding open and phosphorylated complexes (Beacham et al., 2018). Here, we report high-resolution cryo-EM structures of NECAP bound to phosphorylated AP2. The site of AP2 phosphorylation is directly coordinated by residues of the NECAP PHear domain that are predicted from genetic screens in . Using membrane mimetics to generate conformationally open AP2, we find that a second domain of NECAP binds these complexes and cryo-EM reveals both domains of NECAP engaging closed, inactive AP2. Assays in vitro and in vivo confirm these domains cooperate to inactivate AP2. We propose that phosphorylation marks adaptors for inactivation. | ||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6owo.cif.gz | 358.1 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6owo.ent.gz | 293.9 KB | Display | PDB format |
PDBx/mmJSON format | 6owo.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6owo_validation.pdf.gz | 1.2 MB | Display | wwPDB validaton report |
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Full document | 6owo_full_validation.pdf.gz | 1.2 MB | Display | |
Data in XML | 6owo_validation.xml.gz | 59.8 KB | Display | |
Data in CIF | 6owo_validation.cif.gz | 91 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ow/6owo ftp://data.pdbj.org/pub/pdb/validation_reports/ow/6owo | HTTPS FTP |
-Related structure data
Related structure data | 20215MC 6oxlC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 69656.297 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ap2a2, Adtab / Plasmid: PACYC_DUET / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P17427*PLUS |
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#2: Protein | Mass: 66953.195 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ap2b1, Clapb1 / Plasmid: PET_DUET / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9DBG3 |
#3: Protein | Mass: 49806.621 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ap2m1, Clapm1 / Plasmid: PET_DUET / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P84091 |
#4: Protein | Mass: 17038.688 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Ap2s1, Ap17, Claps2 / Plasmid: PACYC_DUET / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P62744 |
#5: Protein | Mass: 28629.941 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Mus musculus (house mouse) / Gene: Necap2 / Plasmid: PET-21B / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9D1J1 |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: PHOSPHORYLATED AP2-NECAP CO-COMPLEX / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES |
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Molecular weight | Experimental value: NO |
Source (natural) | Organism: Mus musculus (house mouse) |
Source (recombinant) | Organism: Escherichia coli (E. coli) / Strain: BL21(DE3) |
Buffer solution | pH: 8 |
Specimen | Conc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Details: GLOW DISCHARGE / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K Details: 4 UL OF SAMPLE/GRID WAS BLOTTED FOR 4 SECONDS AND PLUNGE FROZEN IN LIQUID-NITROGEN COOLED ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
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Microscopy | Model: FEI TALOS ARCTICA |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 36000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm |
Specimen holder | Cryogen: NITROGEN |
Image recording | Electron dose: 1 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 944 |
-Processing
EM software |
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Image processing |
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CTF correction |
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Symmetry |
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3D reconstruction |
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Atomic model building | B value: 63 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: MAXIMUM LIKELIHOOD Details: STARTING MODEL WAS GENERATED BY DOCKING PDB ENTRIES 2VGL AND 1TQZ INTO CRYO-EM DENSITY AND MANUALLY EDITING SEQUENCE AND STRUCTURAl CHANGES. MODEL REFINEMENT WAS PERFORMED USING ROSETTA AND ...Details: STARTING MODEL WAS GENERATED BY DOCKING PDB ENTRIES 2VGL AND 1TQZ INTO CRYO-EM DENSITY AND MANUALLY EDITING SEQUENCE AND STRUCTURAl CHANGES. MODEL REFINEMENT WAS PERFORMED USING ROSETTA AND PHENIX.REAL_SPACE_REFINE | ||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building |
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Refinement | Highest resolution: 3.2 Å |