+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-24712 | |||||||||
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Title | AP2 bound to heparin and Tgn38 tyrosine cargo peptide | |||||||||
Map data | sharpened map | |||||||||
Sample |
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Keywords | AP2 / clathrin vesicle / endocytosis / lipid-binding / adaptor / membrane / transport / cargo | |||||||||
Function / homology | Function and homology information Formation of annular gap junctions / Gap junction degradation / LDL clearance / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / WNT5A-dependent internalization of FZD4 / Trafficking of GluR2-containing AMPA receptors / Retrograde neurotrophin signalling / clathrin adaptor complex / clathrin coat / extrinsic component of presynaptic endocytic zone membrane ...Formation of annular gap junctions / Gap junction degradation / LDL clearance / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / WNT5A-dependent internalization of FZD4 / Trafficking of GluR2-containing AMPA receptors / Retrograde neurotrophin signalling / clathrin adaptor complex / clathrin coat / extrinsic component of presynaptic endocytic zone membrane / VLDLR internalisation and degradation / cardiac septum development / Recycling pathway of L1 / AP-2 adaptor complex / regulation of vesicle size / postsynaptic neurotransmitter receptor internalization / clathrin coat assembly / Cargo recognition for clathrin-mediated endocytosis / positive regulation of synaptic vesicle endocytosis / clathrin adaptor activity / Clathrin-mediated endocytosis / membrane coat / vesicle budding from membrane / clathrin-dependent endocytosis / MHC class II antigen presentation / coronary vasculature development / positive regulation of protein localization to membrane / neurotransmitter receptor internalization / 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 / 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 / kinase binding / intracellular protein transport / cytoplasmic side of plasma membrane / disordered domain specific binding / 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 Similarity search - Function | |||||||||
Biological species | Mus musculus (house mouse) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.7 Å | |||||||||
Authors | Baker RW / Hollopeter G | |||||||||
Funding support | United States, 1 items
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Citation | Journal: Nat Struct Mol Biol / Year: 2022 Title: Structural basis of an endocytic checkpoint that primes the AP2 clathrin adaptor for cargo internalization. Authors: Edward A Partlow / Kevin S Cannon / Gunther Hollopeter / Richard W Baker / Abstract: Clathrin-mediated endocytosis (CME) is the main route of internalization from the plasma membrane. It is known that the heterotetrameric AP2 clathrin adaptor must open to simultaneously engage ...Clathrin-mediated endocytosis (CME) is the main route of internalization from the plasma membrane. It is known that the heterotetrameric AP2 clathrin adaptor must open to simultaneously engage membrane and endocytic cargo, yet it is unclear how transmembrane cargos are captured to catalyze CME. Using cryogenic-electron microscopy, we discover a new way in which mouse AP2 can reorganize to expose membrane- and cargo-binding pockets, which is not observed in clathrin-coated structures. Instead, it is stimulated by endocytic pioneer proteins called muniscins, which do not enter vesicles. Muniscin-engaged AP2 is primed to rearrange into the vesicle-competent conformation on binding the tyrosine cargo internalization motif (YxxΦ). We propose adaptor priming as a checkpoint to ensure cargo internalization. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_24712.map.gz | 97.2 MB | EMDB map data format | |
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Header (meta data) | emd-24712-v30.xml emd-24712.xml | 26.9 KB 26.9 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_24712_fsc.xml | 13.8 KB | Display | FSC data file |
Images | emd_24712.png | 56.9 KB | ||
Masks | emd_24712_msk_1.map | 103 MB | Mask map | |
Filedesc metadata | emd-24712.cif.gz | 7.6 KB | ||
Others | emd_24712_additional_1.map.gz emd_24712_additional_2.map.gz emd_24712_half_map_1.map.gz emd_24712_half_map_2.map.gz | 4 MB 4.2 MB 95.3 MB 95.3 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-24712 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-24712 | HTTPS FTP |
-Validation report
Summary document | emd_24712_validation.pdf.gz | 981 KB | Display | EMDB validaton report |
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Full document | emd_24712_full_validation.pdf.gz | 980.5 KB | Display | |
Data in XML | emd_24712_validation.xml.gz | 18.3 KB | Display | |
Data in CIF | emd_24712_validation.cif.gz | 23.7 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-24712 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-24712 | HTTPS FTP |
-Related structure data
Related structure data | 7rwaMC 7rw8C 7rw9C 7rwbC 7rwcC C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_24712.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | sharpened map | ||||||||||||||||||||
Voxel size | X=Y=Z: 1.155 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_24712_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: local resolution file
File | emd_24712_additional_1.map | ||||||||||||
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Annotation | local resolution file | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: map filtered my local resolution
File | emd_24712_additional_2.map | ||||||||||||
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Annotation | map filtered my local resolution | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half map A
File | emd_24712_half_map_1.map | ||||||||||||
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Annotation | half map A | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half map B
File | emd_24712_half_map_2.map | ||||||||||||
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Annotation | half map B | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : AP2 bound to heparin and Tgn38 tyrosine cargo peptide
Entire | Name: AP2 bound to heparin and Tgn38 tyrosine cargo peptide |
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Components |
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-Supramolecule #1: AP2 bound to heparin and Tgn38 tyrosine cargo peptide
Supramolecule | Name: AP2 bound to heparin and Tgn38 tyrosine cargo peptide / 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 Details: purified with a C-terminal GST tag, removed with PreScission Protease Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Mus musculus (house mouse) |
Molecular weight | Theoretical: 70.57332 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MPAVSKGDGM RGLAVFISDI RNCKSKEAEI KRINKELANI RSKFKGDKAL DGYSKKKYVC KLLFIFLLGH DIDFGHMEAV NLLSSNRYT EKQIGYLFIS VLVNSNSELI RLINNAIKND LASRNPTFMG LALHCIANVG SREMAEAFAG EIPKILVAGD T MDSVKQSA ...String: MPAVSKGDGM 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 AKLKKKKGGS GLEVLFQ UniProtKB: AP-2 complex subunit alpha-2 |
-Macromolecule #2: AP-2 complex subunit beta
Macromolecule | Name: AP-2 complex subunit beta / type: protein_or_peptide / ID: 2 / Number of copies: 2 / 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 UniProtKB: AP-2 complex subunit beta |
-Macromolecule #3: AP-2 complex subunit mu
Macromolecule | Name: AP-2 complex subunit mu / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Mus musculus (house mouse) |
Molecular weight | Theoretical: 49.726641 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 QITSQVTGQI G WRREGIKY ...String: MIGGLFIYNH KGEVLISRVY RDDIGRNAVD AFRVNVIHAR QQVRSPVTNI ARTSFFHVKR SNIWLAAVTK QNVNAAMVFE FLYKMCDVM AAYFGKISEE NIKNNFVLIY ELLDEILDFG YPQNSETGAL KTFITQQGIK SQHQTKEEQS QITSQVTGQI G WRREGIKY RRNELFLDVL ESVNLLMSPQ GQVLSAHVSG RVVMKSYLSG MPECKFGMND KIVIEKQGKG TADETSKSGK QS IAIDDCT FHQCVRLSKF DSERSISFIP PDGEFELMRY RTTKDIILPF RVIPLVREVG RTKLEVKVVI KSNFKPSLLA QKI EVRIPT PLNTSGVQVI CMKGKAKYKA SENAIVWKIK RMAGMKESQI SAEIELLPTN DKKKWARPPI SMNFEVPFAP SGLK VRYLK VFEPKLNYSD HDVIKWVRYI GRSGIYETRC UniProtKB: AP-2 complex subunit mu |
-Macromolecule #4: AP-2 complex subunit sigma
Macromolecule | Name: AP-2 complex subunit sigma / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Mus musculus (house mouse) |
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 UniProtKB: AP-2 complex subunit sigma |
-Macromolecule #5: Trans-Golgi network integral membrane protein TGN38 peptide
Macromolecule | Name: Trans-Golgi network integral membrane protein TGN38 peptide type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Mus musculus (house mouse) |
Molecular weight | Theoretical: 1.810152 KDa |
Sequence | String: CKVTRRPKAA DYQRL |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 2 mg/mL | ||||||||||||
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Buffer | pH: 7.4 Component:
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Grid | Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: OTHER | ||||||||||||
Vitrification | Cryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Blot force -10, 4 second blot, 4 uL sample. | ||||||||||||
Details | Heparin added at a 2-fold excess 20 mM HEPES pH 7.4, 150 mM NaCl, 1 mM DTT, 0.05% beta-OG |
-Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 55.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 36000 |
Sample stage | Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |