+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-20215 | |||||||||
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Title | CRYO-EM STRUCTURE OF PHOSPHORYLATED AP-2 CORE BOUND TO NECAP | |||||||||
Map data | cryo-EM structure of phosphorylated AP2 bound to NECAP; sharpened map | |||||||||
Sample |
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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 / clathrin coat assembly / Cargo recognition for clathrin-mediated endocytosis / Cargo recognition for clathrin-mediated endocytosis / Clathrin-mediated endocytosis / clathrin adaptor activity / Clathrin-mediated endocytosis / positive regulation of synaptic vesicle endocytosis / membrane coat / vesicle budding from membrane / clathrin-dependent endocytosis / coronary vasculature development / MHC class II antigen presentation / positive regulation of protein localization to membrane / neurotransmitter receptor internalization / signal sequence binding / aorta development / 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 receptor internalization / positive regulation of endocytosis / synaptic vesicle endocytosis / negative regulation of protein localization to plasma membrane / protein serine/threonine kinase binding / vesicle-mediated transport / Neutrophil degranulation / phosphatidylinositol binding / secretory granule / kidney development / intracellular protein transport / kinase binding / cytoplasmic side of plasma membrane / endocytosis / disordered domain specific binding / protein transport / synaptic vesicle / heart development / cytoplasmic vesicle / protein-containing complex assembly / transmembrane transporter binding / postsynapse / protein domain specific binding / intracellular membrane-bounded organelle / lipid binding / glutamatergic synapse / 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 | single particle reconstruction / cryo EM / Resolution: 3.2 Å | |||||||||
Authors | Partlow EA / Baker RW / Beacham GM / Chappie JS / Leschziner AE / Hollopeter G | |||||||||
Funding support | United States, 1 items
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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 | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_20215.map.gz | 96.6 MB | EMDB map data format | |
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Header (meta data) | emd-20215-v30.xml emd-20215.xml | 25.8 KB 25.8 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_20215_fsc.xml | 12.5 KB | Display | FSC data file |
Images | emd_20215.png | 55 KB | ||
Others | emd_20215_additional.map.gz emd_20215_half_map_1.map.gz emd_20215_half_map_2.map.gz | 80.9 MB 80.9 MB 80.8 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-20215 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-20215 | HTTPS FTP |
-Validation report
Summary document | emd_20215_validation.pdf.gz | 847.3 KB | Display | EMDB validaton report |
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Full document | emd_20215_full_validation.pdf.gz | 846.9 KB | Display | |
Data in XML | emd_20215_validation.xml.gz | 17.7 KB | Display | |
Data in CIF | emd_20215_validation.cif.gz | 23.3 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-20215 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-20215 | HTTPS FTP |
-Related structure data
Related structure data | 6owoMC 6oxlC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_20215.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | cryo-EM structure of phosphorylated AP2 bound to NECAP; sharpened map | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.16 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Additional map: cryo-EM structure of phosphorylated AP2 bound to NECAP;...
File | emd_20215_additional.map | ||||||||||||
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Annotation | cryo-EM structure of phosphorylated AP2 bound to NECAP; unsharpened map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: cryo-EM structure of phosphorylated AP2 bound to NECAP;...
File | emd_20215_half_map_1.map | ||||||||||||
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Annotation | cryo-EM structure of phosphorylated AP2 bound to NECAP; unfiltered half-map 1 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: cryo-EM structure of phosphorylated AP2 bound to NECAP;...
File | emd_20215_half_map_2.map | ||||||||||||
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Annotation | cryo-EM structure of phosphorylated AP2 bound to NECAP; unfiltered half-map 2 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : PHOSPHORYLATED AP2-NECAP CO-COMPLEX
Entire | Name: PHOSPHORYLATED AP2-NECAP CO-COMPLEX |
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Components |
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-Supramolecule #1: PHOSPHORYLATED AP2-NECAP CO-COMPLEX
Supramolecule | Name: PHOSPHORYLATED AP2-NECAP CO-COMPLEX / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: Mus musculus (house mouse) |
-Macromolecule #1: AP-2 complex subunit alpha-2
Macromolecule | Name: AP-2 complex subunit alpha-2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Mus musculus (house mouse) |
Molecular weight | Theoretical: 69.656297 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MPAVSKGEGM RGLAVFISDI RNCKSKEAEI KRINKELANI RSKFKGDKAL DGYSKKKYVC KLLFIFLLGH DIDFGHMEAV NLLSSNRYT EKQIGYLFIS VLVNSNSELI RLINNAIKND LASRNPTFMG LALHCIANVG SREMAEAFAG EIPKILVAGD T MDSVKQSA ...String: MPAVSKGEGM RGLAVFISDI RNCKSKEAEI KRINKELANI RSKFKGDKAL DGYSKKKYVC KLLFIFLLGH DIDFGHMEAV NLLSSNRYT EKQIGYLFIS VLVNSNSELI RLINNAIKND LASRNPTFMG LALHCIANVG SREMAEAFAG EIPKILVAGD T MDSVKQSA ALCLLRLYRT SPDLVPMGDW TSRVVHLLND QHLGVVTAAT SLITTLAQKN PEEFKTSVSL AVSRLSRIVT SA STDLQDY TYYFVPAPWL SVKLLRLLQC YPPPEDPAVR GRLTECLETI LNKAQEPPKS KKVQHSNAKN AVLFEAISLI IHH DSEPNL LVRACNQLGQ FLQHRETNLR YLALESMCTL ASSEFSHEAV KTHIETVINA LKTERDVSVR QRAVDLLYAM CDRS NAQQI VAEMLSYLET ADYSIREEIV LKVAILAEKY AVDYTWYVDT ILNLIRIAGD YVSEEVWYRV IQIVINRDDV QGYAA KTVF EALQAPACHE NLVKVGGYIL GEFGNLIAGD PRSSPLIQFN LLHSKFHLCS VPTRALLLST YIKFVNLFPE VKATIQ DVL RSDSQLKNAD VELQQRAVEY LRLSTVASTD ILATVLEEMP PFPERESSIL AKLKKKKG |
-Macromolecule #2: AP-2 complex subunit beta
Macromolecule | Name: AP-2 complex subunit beta / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Mus musculus (house mouse) |
Molecular weight | Theoretical: 66.953195 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MTDSKYFTTN KKGEIFELKA ELNNEKKEKR KEAVKKVIAA MTVGKDVSSL FPDVVNCMQT DNLELKKLVY LYLMNYAKSQ PDMAIMAVN SFVKDCEDPN PLIRALAVRT MGCIRVDKIT EYLCEPLRKC LKDEDPYVRK TAAVCVAKLH DINAQMVEDQ G FLDSLRDL ...String: MTDSKYFTTN KKGEIFELKA ELNNEKKEKR KEAVKKVIAA MTVGKDVSSL FPDVVNCMQT DNLELKKLVY LYLMNYAKSQ PDMAIMAVN SFVKDCEDPN PLIRALAVRT MGCIRVDKIT EYLCEPLRKC LKDEDPYVRK TAAVCVAKLH DINAQMVEDQ G FLDSLRDL IADSNPMVVA NAVAALSEIS ESHPNSNLLD LNPQNINKLL TALNECTEWG QIFILDCLSN YNPKDDREAQ SI CERVTPR LSHANSAVVL SAVKVLMKFL ELLPKDSDYY NMLLKKLAPP LVTLLSGEPE VQYVALRNIN LIVQKRPEIL KQE IKVFFV KYNDPIYVKL EKLDIMIRLA SQANIAQVLA ELKEYATEVD VDFVRKAVRA IGRCAIKVEQ SAERCVSTLL DLIQ TKVNY VVQEAIVVIR DIFRKYPNKY ESIIATLCEN LDSLDEPDAR AAMIWIVGEY AERIDNADEL LESFLEGFHD ESTQV QLTL LTAIVKLFLK KPSETQELVQ QVLSLATQDS DNPDLRDRGY IYWRLLSTDP VTAKEVVLSE KPLISEETDL IEPTLL DEL ICHIGSLASV YHKPPNAFVE GSHGIHRK |
-Macromolecule #3: AP-2 complex subunit mu
Macromolecule | Name: AP-2 complex subunit mu / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Mus musculus (house mouse) |
Molecular weight | Theoretical: 49.806621 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MIGGLFIYNH KGEVLISRVY RDDIGRNAVD AFRVNVIHAR QQVRSPVTNI ARTSFFHVKR SNIWLAAVTK QNVNAAMVFE FLYKMCDVM AAYFGKISEE NIKNNFVLIY ELLDEILDFG YPQNSETGAL KTFITQQGIK SQHQTKEEQS QITSQV(TPO) GQ IGWRREGIKY ...String: MIGGLFIYNH KGEVLISRVY RDDIGRNAVD AFRVNVIHAR QQVRSPVTNI ARTSFFHVKR SNIWLAAVTK QNVNAAMVFE FLYKMCDVM AAYFGKISEE NIKNNFVLIY ELLDEILDFG YPQNSETGAL KTFITQQGIK SQHQTKEEQS QITSQV(TPO) GQ IGWRREGIKY RRNELFLDVL ESVNLLMSPQ GQVLSAHVSG RVVMKSYLSG MPECKFGMND KIVIEKQGKG TADETSKS G KQSIAIDDCT FHQCVRLSKF DSERSISFIP PDGEFELMRY RTTKDIILPF RVIPLVREVG RTKLEVKVVI KSNFKPSLL AQKIEVRIPT PLNTSGVQVI CMKGKAKYKA SENAIVWKIK RMAGMKESQI SAEIELLPTN DKKKWARPPI SMNFEVPFAP SGLKVRYLK VFEPKLNYSD HDVIKWVRYI GRSGIYETRC |
-Macromolecule #4: AP-2 complex subunit sigma
Macromolecule | Name: AP-2 complex subunit sigma / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Rattus norvegicus (Norway rat) |
Molecular weight | Theoretical: 17.038688 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MIRFILIQNR AGKTRLAKWY MQFDDDEKQK LIEEVHAVVT VRDAKHTNFV EFRNFKIIYR RYAGLYFCIC VDVNDNNLAY LEAIHNFVE VLNEYFHNVC ELDLVFNFYK VYTVVDEMFL AGEIRETSQT KVLKQLLMLQ SLE |
-Macromolecule #5: Adaptin ear-binding coat-associated protein 2
Macromolecule | Name: Adaptin ear-binding coat-associated protein 2 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Mus musculus (house mouse) |
Molecular weight | Theoretical: 28.629941 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MEESEYESVL CVKPEVHVYR IPPRATNRGY RASEWQLDQP SWSGRLRITA KGKVAYIKLE DRTSGELFAQ APVDQFPGTA VESVTDSSR YFVIRIEDGN GRRAFIGLGF GDRGDAFDFN VALQDHFKWV KQQCEFAKQA QNPDEGPKLD LGFKDGQTIK I NIANMRKK ...String: MEESEYESVL CVKPEVHVYR IPPRATNRGY RASEWQLDQP SWSGRLRITA KGKVAYIKLE DRTSGELFAQ APVDQFPGTA VESVTDSSR YFVIRIEDGN GRRAFIGLGF GDRGDAFDFN VALQDHFKWV KQQCEFAKQA QNPDEGPKLD LGFKDGQTIK I NIANMRKK EGAAGTPRAR PTSAGGLSLL PPPPGGKSST VIPPSGEQLS VGGSLVQPAV VSGSGGATEL WPQSKPAAAA TA DIWGDFT KSTGSPSSQS QPGTGWVQF |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1.0 mg/mL |
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Buffer | pH: 8 |
Grid | Model: UltrAuFoil / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: GLOW DISCHARGE |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV Details: 4 UL OF SAMPLE/GRID WAS BLOTTED FOR 4 SECONDS AND PLUNGE FROZEN IN LIQUID-NITROGEN COOLED ETHANE. |
-Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Software | Name: Leginon |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number real images: 944 / Average electron dose: 1.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 36000 |
Sample stage | Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
+Image processing #1
+Image processing #2
-Atomic model buiding 1
Initial model | (PDB ID: , ) |
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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 |
Refinement | Space: REAL / Protocol: RIGID BODY FIT / Overall B value: 63 / Target criteria: MAXIMUM LIKELIHOOD |
Output model | PDB-6owo: |