2024-05-22 REL PDBe PDBe PDBe 2020-03-12 2020-07-29 2020-07-29 2024-05-22 Wellcome Trust 207455/Z/17/Z United Kingdom Medical Research Council (MRC, United Kingdom) MC_UP_1201/16 United Kingdom Clathrin with bound beta2 appendage of AP2 Kovtun O Kane Dickson V clathrin, clathrin adaptor, ap2, clathrin assembly, ENDOCYTOSIS Kovtun O Dickson VK Kelly BT Owen DJ Briggs JAG Architecture of the AP2/clathrin coat on the membranes of clathrin-coated vesicles. Sci Adv US 6 eaba8381 eaba8381 2020 32743075 doi:10.1126/sciadv.aba8381 2375-2548 EMD-10747 other EM volume
AP2 core in physiological buffer
 EMD-10748 other EM volume EMD-10749 other EM volume EMD-10750 other EM volume EMD-10751 other EM volume EMD-10752 other EM volume EMD-10753 other EM volume 6yai FULLOVERLAP Clathrin/AP2 coat assembled on membrane. Clathrin/AP2 coat assembled on membrane. 0 1 2 3 4
Clathrin/AP coat was formed on the membrane containing cargo signal peptides. The AP2 adaptor lacked hinge and appendage regions in its alpha subunit.
 Clathrin 1 1 3 4
Clathrin in the clathrin/AP2 coat formed on the membrane
Sus scrofa brain beta2 appendage of AP2 adaptor 1 2
beta2 appendage of the AP2 bound to clathrin to clathrin/AP2 coat formed on the membrane
Homo sapiens Clathrin heavy chain Sus scrofa 0.187145125 8 LEVO MAQILPIRFQEHLQLQNLGINPANIGFSTLTMESDKFICIREKVGEQAQVVIIDMNDPSNPIRRPISADSAIMNPASKVI ALKAGKTLQIFNIEMKSKMKAHTMTDDVTFWKWISLNTVALVTDNAVYHWSMEGESQPVKMFDRHSSLAGCQIINYRTDA KQKWLLLTGISAQQNRVVGAMQLYSVDRKVSQPIEGHAASFAQFKMEGNAEESTLFCFAVRGQAGGKLHIIEVGTPPTGN QPFPKKAVDVFFPPEAQNDFPVAMQISEKHDVVFLITKYGYIHLYDLETGTCIYMNRISGETIFVTAPHEATAGIIGVNR KGQVLSVCVEEENIIPYITNVLQNPDLALRMAVRNNLAGAEELFARKFNALFAQGNYSEAAKVAANAPKGILRTPDTIRR FQSVPAQPGQTSPLLQYFGILLDQGQLNKYESLELCRPVLQQGRKQLLEKWLKEDKLECSEELGDLVKSVDPTLALSVYL RANVPNKVIQCFAETGQVQKIVLYAKKVGYTPDWIFLLRNVMRISPDQGQQFAQMLVQDEEPLADITQIVDVFMEYNLIQ QCTAFLLDALKNNRPSEGPLQTRLLEMNLMHAPQVADAILGNQMFTHYDRAHIAQLCEKAGLLQRALEHFTDLYDIKRAV VHTHLLNPEWLVNYFGSLSVEDSLECLRAMLSANIRQNLQICVQVASKYHEQLSTQSLIELFESFKSFEGLFYFLGSIVN FSQDPDVHFKYIQAACKTGQIKEVERICRESNCYDPERVKNFLKEAKLTDQLPLIIVCDRFDFVHDLVLYLYRNNLQKYI EIYVQKVNPSRLPVVIGGLLDVDCSEDVIKNLILVVRGQFSTDELVAEVEKRNRLKLLLPWLEARIHEGCEEPATHNALA KIYIDSNNNPERFLRENPYYDSRVVGKYCEKRDPHLACVAYERGQCDLELINVCNENSLFKSLSRYLVRRKDPELWGSVL LESNPYRRPLIDQVVQTALSETQDPEEVSVTVKAFMTADLPNELIELLEKIVLDNSVFSEHRNLQNLLILTAIKADRTRV MEYINRLDNYDAPDIANIAISNELFEEAFAIFRKFDVNTSAVQVLIEHIGNLDRAYEFAERCNEPAVWSQLAKAQLQKGM VKEAIDSYIKADDPSSYMEVVQAANTSGNWEELVKYLQMARKKARESYVETELIFALAKTNRLAELEEFINGPNNAHIQQ VGDRCYDEKMYDAAKLLYNNVSNFGRLASTLVHLGEYQAAVDGARKANSTRTWKEVCFACVDGKEFRLAQMCGLHIVVHA DELEELINYYQDRGYFEELITMLEAALGLERAHMGMFTELAILYSKFKPQKMREHLELFWSRVNIPKVLRAAEQAHLWAE LVFLYDKYEEYDNAIITMMNHPTDAWKEGQFKDIITKVANVELYYRAIQFYLEFKPLLLNDLLMVLSPRLDHTRAVNYFS KVKQLPLVKPYLRSVQNHNNKSVNESLNNLFITEEDYQALRTSIDAYDNFDNISLAQRLEKHELIEFRRIAAYLFKGNNR WKQSVELCKKDSLYKDAMQYASESKDTELAEELLQWFLQEEKRECFGACLFTCYDLLRPDVVLETAWRHNIMDFAMPYFI QVMKEYLTKVDKLDASESLRKEEEQATETQ C0MHR2 AP-2 complex subunit beta Homo sapiens 0.026429456999999996 1 Escherichia coli 'BL21-Gold(DE3)pLysS AG' LEVO GGYVAPKAVWLPAVKAKGLEISGTFTHRQGHIYMEMNFTNKALQHMTDFAIQFNKNSFGVIPSTPLAIHTPLMPNQSIDV SLPLNTLGPVMKMEPLNNLQVAVKNNIDVFYFSCLIPLNVLFVEDGKMERQVFLATWKDIPNENELQFQIKECHLNADTV SSKLQNNNVYTIAKRNVEGQDMLYQSLKLTNGIWILAELRIQPGNPNYTLSLKCRAPEVSQYIYQVYDSILKN P63010 Clathrin heavy chain Sus scrofa 0.187117094 1 LEVO MAQILPIRFQEHLQLQNLGINPANIGFSTLTMESDKFICIREKVGEQAQVVIIDMNDPSNPIRRPISADSAIMNPASKVI ALKAGKTLQIFNIEMKSKMKAHTMTDDVTFWKWISLNTVALVTDNAVYHWSMEGESQPVKMFDRHSSLAGCQIINYRTDA KQKWLLLTGISAQQNRVVGAMQLYSVDRKVSQPIEGHAASFAQFKMEGNAEESTLFCFAVRGQAGGKLHIIEVGTPPTGN QPFPKKAVDVFFPPEAQNDFPVAMQISEKHDVVFLITKYGYIHLYDLETGTCIYMNRISGETIFVTAPHEATAGIIGVNR KGQVLSVCVEEENIIPYITNVLQNPDLALRMAVRNNLAGAEELFARKFNALFAQGNYSEAAKVAANAPKGILRTPDTIRR FQSVPAQPGQTSPLLQYFGILLDQGQLNKYESLELCRPVLQQGRKQLLEKWLKEDKLECSEELGDLVKSVDPTLALSVYL RANVPNKVIQCFAETGQVQKIVLYAKKVGYTPDWIFLLRNVMRISPDQGQQFAQMLVQDEEPLADITQIVDVFMEYNLIQ QCTAFLLDALKNNRPSEGPLQTRLLEMNLMHAPQVADAILGNQMFTHYDRAHIAQLCEKAGLLQRALEHFTDLYDIKRAV VHTHLLNPEWLVNYFGSLSVEDSLECLRAMLSANIRQNLQICVQVASKYHEQLSTQSLIELFESFKSFEGLFYFLGSIVN FSQDPDVHFKYIQAACKTGQIKEVERICRESNCYDPERVKNFLKEAKLTDQLPLIIVCDRFDFVHDLVLYLYRNNLQKYI EIYVQKVNPSRLPVVIGGLLDVDCSEDVIKNLILVVRGQFSTDELVAEVEKRNRLKLLLPWLEARIHEGCTEPATHNALA KIYIDSNNNPERFLRENPYYDSRVVGKYCEKRDPHLACVAYERGQCDLELINVCNENSLFKSLSRYLVRRKDPELWGSVL LESNPYRRPLIDQVVQTALSETQDPEEVSVTVKAFMTADLPNELIELLEKIVLDNSVFSEHRNLQNLLILTAIKADRTRV MEYINRLDNYDAPDIANIAISNELFEEAFAIFRKFDVNTSAVQVLIEHIGNLDRAYEFAERCNEPAVWSQLAKAQLQKGM VKEAIDSYIKADDPSSYMEVVQAANTSGNWEELVKYLQMARKKARESYVETELIFALAKTNRLAELEEFINGPNNAHIQQ VGDRCYDEKMYDAAKLLYNNVSNFGRLASTLVHLGEYQAAVDGARKANSTRTWKEVCFACVDGKEFRLAQMCGLHIVVHA DELEELINYYQDRGYFEELITMLEAALGLERAHMGMFTELAILYSKFKPQKMREHLELFWSRVNIPKVLRAAEQAHLWAE LVFLYDKYEEYDNAIITMMNHPTDAWKEGQFKDIITKVANVELYYRAIQFYLEFKPLLLNDLLMVLSPRLDHTRAVNYFS KVKQLPLVKPYLRSVQNHNNKSVNESLNNLFITEEDYQALRTSIDAYDNFDNISLAQRLEKHELIEFRRIAAYLFKGNNR WKQSVELCKKDSLYKDAMQYASESKDTELAEELLQWFLQEEKRECFGACLFTCYDLLRPDVVLETAWRHNIMDFAMPYFI QVMKEYLTKVDKLDASESLRKEEEQATETQ C0MHR2 Clathrin light chain Sus scrofa 0.025218499999999998 4 LEVO MADDFGFFSSSESGAPEVAEEDPAAAFLAQQESEIAGIENDEGFGAPAGSQAALAQPGPASGAGPEDMGTTVNGDVFQDA NGPADGYAAIAQADRLTQEPESIRKWREEQRKRLQELDAASKVTEQEWREKAKKDLEEWNQRQSEQVEKNKINNRIADKA FYQQPDADIIGYVASEEAFVKESKEETPGTEWEKVAQLCDFNPKSSKQCKDVSRLRSVLMSLKQTPLSR F1S398 subtomogramAveraging threeDArray 0.7 7.2 10.0 Hepes KOH 120.0 Potassium Acetate 2.0 Magnesium chloride 5.0 2-Mercaptoethanol C-flat-2/2 CARBON HOLEY ETHANE 98 291 LEICA EM GP
The sample was supplemented with 10 nm nanogold fiducials, and 3 ul of the mixture was backside blotted for 3 seconds..
The sample (in vitro budding reaction) contained AP2, clathrin and 400 nm extruded liposomes
 FEI TITAN KRIOS FLOOD BEAM BRIGHT FIELD FIELD EMISSION GUN 300 100.0 2.7 1.5 1.5 6.5 6.5 81000.0 81000.0 FEI TITAN KRIOS AUTOGRID HOLDER NITROGEN GIF Quantum LS 20 GATAN K2 QUANTUM (4k x 4k) COUNTING 3838 3710 1-10 2 0.2 3.2
The images were collected in movie mode at 10 frames per second
 1
The images were low pass filtered according to the cumulative radiation dose.
C1 9.2 FSC 0.143 CUT-OFF TOM AV3 12076 58121 198871 reference-free geometrically defined initial positions TOM
To define initial positions of subtomograms, centres and radii of coated vesicles, buds were manually marked in bin4 tomograms. These measurements were used to define spheres. Subtomogram positions were then defined on the surface of these spheres with uniform sampling. The orientations were calculated to be normal to the surfaces of the spheres with random in-plane rotation.
 10 1200.0 PEET Dynamo
Principal component analysis (PCA) to sort subtomograms by beta-appendage occupancy. The PCA was performed on wedge-masked difference maps with calculations implemented in MATLAB using code adapted from PEET and Dynamo packages.
 OTHER TOM AV3 emd_10754.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 160 160 160 0 0 0 160 160 160 284.8 284.8 284.8 90.0 90.0 90.0 X Y Z -0.44512126 0.6409551 -0.00006332948 0.055856194 1.78 1.78 1.78 0.07 AUTHOR Sharpened map of hexagon bounding clathrin leg enriched in beta-appendage 1XI4 PDB experimental model 6SCT PDB experimental model 1E42 A PDB experimental model FLEXIBLE FIT correlation coefficient REAL emd_10754_msk_1.map emd_10754_half_map_1.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 160 160 160 0 0 0 160 160 160 284.8 284.8 284.8 90.0 90.0 90.0 X Y Z -0.2536598 0.2809683 -0.00014885237 0.03586526 1.78 1.78 1.78 AUTHOR Unsharpened half-map of hexagon bounding clathrin leg enriched in beta2-appendage emd_10754_half_map_2.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 160 160 160 0 0 0 160 160 160 284.8 284.8 284.8 90.0 90.0 90.0 X Y Z -0.2528162 0.3071754 -0.00014692369 0.040701315 1.78 1.78 1.78 AUTHOR Unsharpened half-map of hexagon bounding clathrin leg enriched in beta-appendage