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Yorodumi- EMDB-10751: AP2 in clathrin coats assembled on a membrane containing dileucin... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-10751 | |||||||||
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Title | AP2 in clathrin coats assembled on a membrane containing dileucine- and tyrosine-based cargo peptides | |||||||||
Map data | Sharpened map of AP2 in clathrin coat formed on membranes containing dileucine- and tyrosine-based cargo peptides | |||||||||
Sample |
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Keywords | clathrin / clathrin adaptor / ap2 / clathrin assembly / ENDOCYTOSIS | |||||||||
Function / homology | Function and homology information Gap junction degradation / Formation of annular gap junctions / 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 / Nef Mediated CD8 Down-regulation ...Gap junction degradation / Formation of annular gap junctions / 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 / Nef Mediated CD8 Down-regulation / Trafficking of GluR2-containing AMPA receptors / Retrograde neurotrophin signalling / clathrin adaptor complex / WNT5A-dependent internalization of FZD4 / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / Trafficking of GluR2-containing AMPA receptors / extrinsic component of presynaptic endocytic zone membrane / WNT5A-dependent internalization of FZD4 / VLDLR internalisation and degradation / 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 / Retrograde neurotrophin signalling / Cargo recognition for clathrin-mediated endocytosis / positive regulation of synaptic vesicle endocytosis / clathrin-coated endocytic vesicle / LDL clearance / Clathrin-mediated endocytosis / clathrin adaptor activity / Clathrin-mediated endocytosis / membrane coat / vesicle budding from membrane / clathrin-dependent endocytosis / MHC class II antigen presentation / coronary vasculature development / signal sequence binding / Nef Mediated CD4 Down-regulation / endolysosome membrane / aorta development / negative regulation of protein localization to plasma membrane / ventricular septum development / Neutrophil degranulation / low-density lipoprotein particle receptor binding / clathrin binding / Recycling pathway of L1 / Trafficking of GluR2-containing AMPA receptors / positive regulation of receptor internalization / synaptic vesicle endocytosis / EPH-ephrin mediated repulsion of cells / protein serine/threonine kinase binding / clathrin-coated pit / vesicle-mediated transport / MHC class II antigen presentation / VLDLR internalisation and degradation / phosphatidylinositol binding / kidney development / intracellular protein transport / clathrin-coated endocytic vesicle membrane / terminal bouton / receptor internalization / cytoplasmic side of plasma membrane / kinase binding / disordered domain specific binding / endocytic vesicle membrane / Cargo recognition for clathrin-mediated endocytosis / synaptic vesicle / Clathrin-mediated endocytosis / presynapse / cytoplasmic vesicle / protein-containing complex assembly / transmembrane transporter binding / postsynapse / Potential therapeutics for SARS / protein domain specific binding / intracellular membrane-bounded organelle / lipid binding / glutamatergic synapse / synapse / protein-containing complex binding / protein kinase binding / membrane / plasma membrane / cytosol Similarity search - Function | |||||||||
Biological species | Rattus norvegicus (Norway rat) / Homo sapiens (human) / Mus musculus (house mouse) | |||||||||
Method | subtomogram averaging / cryo EM / Resolution: 10.2 Å | |||||||||
Authors | Kovtun O / Kane Dickson V | |||||||||
Funding support | United Kingdom, 2 items
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Citation | Journal: Sci Adv / Year: 2020 Title: Architecture of the AP2/clathrin coat on the membranes of clathrin-coated vesicles. Authors: Oleksiy Kovtun / Veronica Kane Dickson / Bernard T Kelly / David J Owen / John A G Briggs / Abstract: Clathrin-mediated endocytosis (CME) is crucial for modulating the protein composition of a cell's plasma membrane. Clathrin forms a cage-like, polyhedral outer scaffold around a vesicle, to which ...Clathrin-mediated endocytosis (CME) is crucial for modulating the protein composition of a cell's plasma membrane. Clathrin forms a cage-like, polyhedral outer scaffold around a vesicle, to which cargo-selecting clathrin adaptors are attached. Adaptor protein complex (AP2) is the key adaptor in CME. Crystallography has shown AP2 to adopt a range of conformations. Here, we used cryo-electron microscopy, tomography, and subtomogram averaging to determine structures, interactions, and arrangements of clathrin and AP2 at the key steps of coat assembly, from AP2 in solution to membrane-assembled clathrin-coated vesicles (CCVs). AP2 binds cargo and PtdIns(4,5) (phosphatidylinositol 4,5-bisphosphate)-containing membranes via multiple interfaces, undergoing conformational rearrangement from its cytosolic state. The binding mode of AP2 β2 appendage into the clathrin lattice in CCVs and buds implies how the adaptor structurally modulates coat curvature and coat disassembly. | |||||||||
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_10751.map.gz | 1.6 MB | EMDB map data format | |
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Header (meta data) | emd-10751-v30.xml emd-10751.xml | 28.2 KB 28.2 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_10751_fsc.xml | 4.4 KB | Display | FSC data file |
Images | emd_10751.png | 124.3 KB | ||
Masks | emd_10751_msk_1.map | 6.6 MB | Mask map | |
Filedesc metadata | emd-10751.cif.gz | 8.2 KB | ||
Others | emd_10751_half_map_1.map.gz emd_10751_half_map_2.map.gz | 6 MB 6.1 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-10751 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-10751 | HTTPS FTP |
-Related structure data
Related structure data | 6yahMC 6yaeC 6yafC 6yaiC C: citing same article (ref.) M: atomic model generated by this map |
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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_10751.map.gz / Format: CCP4 / Size: 6.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Sharpened map of AP2 in clathrin coat formed on membranes containing dileucine- and tyrosine-based cargo peptides | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.787 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Mask #1
File | emd_10751_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: Unsharpened half-map of AP2 in clathrin coat formed...
File | emd_10751_half_map_1.map | ||||||||||||
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Annotation | Unsharpened half-map of AP2 in clathrin coat formed on membranes containing dileucine- and tyrosine-based cargo peptides | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Unsharpened half-map of AP2 in clathrin coat formed...
File | emd_10751_half_map_2.map | ||||||||||||
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Annotation | Unsharpened half-map of AP2 in clathrin coat formed on membranes containing dileucine- and tyrosine-based cargo peptides | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Clathrin/AP2 coat assembled on AP2 membrane.
Entire | Name: Clathrin/AP2 coat assembled on AP2 membrane. |
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Components |
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-Supramolecule #1: Clathrin/AP2 coat assembled on AP2 membrane.
Supramolecule | Name: Clathrin/AP2 coat assembled on AP2 membrane. / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all Details: Clathrin/AP coat was formed on the membrane containing dileucine- and tyrosine-based cargo signal peptides. The AP2 adaptor lacked hinge and appendage regions in its alpha subunit. |
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-Supramolecule #2: AP-2 complex in clathrin coat, subunits alpha-2 and mu
Supramolecule | Name: AP-2 complex in clathrin coat, subunits alpha-2 and mu type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1, #3 Details: AP2 in clathrin coat formed on membranes containing dileucine- and tyrosine-based cargo peptides |
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Source (natural) | Organism: Rattus norvegicus (Norway rat) |
-Supramolecule #3: AP-2 complex in clathrin coat, subunit beta
Supramolecule | Name: AP-2 complex in clathrin coat, subunit beta / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2 Details: AP2 in clathrin coat formed on membranes containing dileucine- and tyrosine-based cargo peptides |
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Source (natural) | Organism: Homo sapiens (human) |
-Supramolecule #4: AP-2 complex in clathrin coat, subunit sigma
Supramolecule | Name: AP-2 complex in clathrin coat, subunit sigma / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #4 Details: AP2 in clathrin coat formed on membranes containing dileucine- and tyrosine-based cargo peptides |
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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: Rattus norvegicus (Norway rat) |
Molecular weight | Theoretical: 70.310062 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
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 GLVPR 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: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 105.619344 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MHHHHHHMTD SKYFTTNKKG EIFELKAELN NEKKEKRKEA VKKVIAAMTV GKDVSSLFPD VVNCMQTDNL ELKKLVYLYL MNYAKSQPD MAIMAVNSFV KDCEDPNPLI RALAVRTMGC IRVDKITEYL CEPLRKCLKD EDPYVRKTAA VCVAKLHDIN A QMVEDQGF ...String: MHHHHHHMTD SKYFTTNKKG EIFELKAELN NEKKEKRKEA VKKVIAAMTV GKDVSSLFPD VVNCMQTDNL ELKKLVYLYL MNYAKSQPD MAIMAVNSFV KDCEDPNPLI RALAVRTMGC IRVDKITEYL CEPLRKCLKD EDPYVRKTAA VCVAKLHDIN A QMVEDQGF LDSLRDLIAD SNPMVVANAV AALSEISESH PNSNLLDLNP QNINKLLTAL NECTEWGQIF ILDCLSNYNP KD DREAQSI CERVTPRLSH ANSAVVLSAV KVLMKFLELL PKDSDYYNML LKKLAPPLVT LLSGEPEVQY VALRNINLIV QKR PEILKQ EIKVFFVKYN DPIYVKLEKL DIMIRLASQA NIAQVLAELK EYATEVDVDF VRKAVRAIGR CAIKVEQSAE RCVS TLLDL IQTKVNYVVQ EAIVVIRDIF RKYPNKYESI IATLCENLDS LDEPDARAAM IWIVGEYAER IDNADELLES FLEGF HDES TQVQLTLLTA IVKLFLKKPS ETQELVQQVL SLATQDSDNP DLRDRGYIYW RLLSTDPVTA KEVVLSEKPL ISEETD LIE PTLLDELICH IGSLASVYHK PPNAFVEGSH GIHRKHLPIH HGSTDAGDSP VGTTTATNLE QPQVIPSQGD LLGDLLN LD LGPPVNVPQV SSMQMGAVDL LGGGLDSLVG QSFIPSSVPA TFAPSPTPAV VSSGLNDLFE LSTGIGMAPG GYVAPKAV W LPAVKAKGLE ISGTFTHRQG HIYMEMNFTN KALQHMTDFA IQFNKNSFGV IPSTPLAIHT PLMPNQSIDV SLPLNTLGP VMKMEPLNNL QVAVKNNIDV FYFSCLIPLN VLFVEDGKME RQVFLATWKD IPNENELQFQ IKECHLNADT VSSKLQNNNV YTIAKRNVE GQDMLYQSLK LTNGIWILAE LRIQPGNPNY TLSLKCRAPE VSQYIYQVYD SILKN 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: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Rattus norvegicus (Norway rat) |
Molecular weight | Theoretical: 51.044113 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
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 TADETSKSME QK LISEEDL GKQSIAIDDC TFHQCVRLSK FDSERSISFI PPDGEFELMR YRTTKDIILP FRVIPLVREV GRTKLEVKVV IKS NFKPSL LAQKIEVRIP TPLNTSGVQV ICMKGKAKYK ASENAIVWKI KRMAGMKESQ ISAEIELLPT NDKKKWARPP ISMN FEVPF APSGLKVRYL KVFEPKLNYS DHDVIKWVRY IGRSGIYETR C 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: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Mus musculus (house mouse) |
Molecular weight | Theoretical: 17.038688 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MIRFILIQNR AGKTRLAKWY MQFDDDEKQK LIEEVHAVVT VRDAKHTNFV EFRNFKIIYR RYAGLYFCIC VDVNDNNLAY LEAIHNFVE VLNEYFHNVC ELDLVFNFYK VYTVVDEMFL AGEIRETSQT KVLKQLLMLQ SLE UniProtKB: AP-2 complex subunit sigma |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | subtomogram averaging |
Aggregation state | 3D array |
-Sample preparation
Concentration | 0.7 mg/mL | ||||||||||
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Buffer | pH: 7.2 Component:
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Grid | Model: C-flat-2/2 / Support film - Material: CARBON / Support film - topology: HOLEY | ||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 98 % / Chamber temperature: 291 K / Instrument: LEICA EM GP Details: The sample was supplemented with 10 nm nanogold fiducials, and 3 ul of the mixture was backside blotted for 3 seconds.. | ||||||||||
Details | The sample (in vitro budding reaction) contained AP2, clathrin and 400 nm extruded liposomes |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-10 / Number grids imaged: 2 / Average exposure time: 0.2 sec. / Average electron dose: 3.2 e/Å2 Details: The images were collected in movie mode at 10 frames per second |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 100.0 µm / Calibrated defocus max: 6.5 µm / Calibrated defocus min: 1.5 µm / Calibrated magnification: 81000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 6.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 81000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Initial model | PDB ID: Chain - Source name: PDB / Chain - Initial model type: experimental model |
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Refinement | Space: REAL / Protocol: FLEXIBLE FIT / Target criteria: correlation coefficient |
Output model | PDB-6yah: |