+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-4036 | |||||||||
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Title | Cryo-EM reconstruction of clathrin D6 cages + Hsc70 Delta C | |||||||||
Map data | None | |||||||||
Sample |
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Biological species | Mus musculus (house mouse) / Bos taurus (cattle) / Rattus norvegicus (Norway rat) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 28.0 Å | |||||||||
Authors | Sousa R / Liao H-S / Cuellar J / Valpuesta JM / Jin AJ / Lafer EM | |||||||||
Citation | Journal: Nat Struct Mol Biol / Year: 2016 Title: Clathrin-coat disassembly illuminates the mechanisms of Hsp70 force generation. Authors: Rui Sousa / Hsien-Shun Liao / Jorge Cuéllar / Suping Jin / José M Valpuesta / Albert J Jin / Eileen M Lafer / Abstract: Hsp70s use ATP hydrolysis to disrupt protein-protein associations and to move macromolecules. One example is the Hsc70- mediated disassembly of the clathrin coats that form on vesicles during ...Hsp70s use ATP hydrolysis to disrupt protein-protein associations and to move macromolecules. One example is the Hsc70- mediated disassembly of the clathrin coats that form on vesicles during endocytosis. Here, we exploited the exceptional features of these coats to test three models-Brownian ratchet, power-stroke and entropic pulling-proposed to explain how Hsp70s transform their substrates. Our data rule out the ratchet and power-stroke models and instead support a collision-pressure mechanism whereby collisions between clathrin-coat walls and Hsc70s drive coats apart. Collision pressure is the complement to the pulling force described in the entropic pulling model. We also found that self-association augments collision pressure, thereby allowing disassembly of clathrin lattices that have been predicted to be resistant to disassembly. These results illuminate how Hsp70s generate the forces that transform their substrates. | |||||||||
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_4036.map.gz | 41.2 MB | EMDB map data format | |
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Header (meta data) | emd-4036-v30.xml emd-4036.xml | 19 KB 19 KB | Display Display | EMDB header |
Images | emd_4036.png | 59.5 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-4036 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-4036 | HTTPS FTP |
-Validation report
Summary document | emd_4036_validation.pdf.gz | 221.9 KB | Display | EMDB validaton report |
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Full document | emd_4036_full_validation.pdf.gz | 221 KB | Display | |
Data in XML | emd_4036_validation.xml.gz | 6.8 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-4036 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-4036 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_4036.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | None | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 3.65 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Cryo-EM reconstruction of clathrin D6 cages + Hsc70 Delta C
Entire | Name: Cryo-EM reconstruction of clathrin D6 cages + Hsc70 Delta C |
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Components |
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-Supramolecule #1: Cryo-EM reconstruction of clathrin D6 cages + Hsc70 Delta C
Supramolecule | Name: Cryo-EM reconstruction of clathrin D6 cages + Hsc70 Delta C type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Molecular weight | Theoretical: 31.476968 MDa |
-Macromolecule #1: AP180
Macromolecule | Name: AP180 / type: protein_or_peptide / ID: 1 / Details: GST tag at N-terminus / Enantiomer: LEVO |
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Source (natural) | Organism: Mus musculus (house mouse) |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MSPILGYWKI KGLVQPTRLL LEYLEEKYEE HLYERDEGDK WRNKKFELGL EFPNLPYYID GDVKLTQSMA IIRYIADKHN MLGGCPKERA EISMLEGAVL DIRYGVSRIA YSKDFETLKV DFLSKLPEML KMFEDRLCHK TYLNGDHVTH PDFMLYDALD VVLYMDPMCL ...String: MSPILGYWKI KGLVQPTRLL LEYLEEKYEE HLYERDEGDK WRNKKFELGL EFPNLPYYID GDVKLTQSMA IIRYIADKHN MLGGCPKERA EISMLEGAVL DIRYGVSRIA YSKDFETLKV DFLSKLPEML KMFEDRLCHK TYLNGDHVTH PDFMLYDALD VVLYMDPMCL DAFPKLVCFK KRIEAIPQID KYLKSSKYIA WPLQGWQATF GGGDHPPKSD LIEGRGIPGS SPATTVTSPN STPAKTIDTS PPVDIFATAS AAAPVSSAKP SSDLLDLQPD FSGAAAGAAA PVVPPSGGAT AWGDLLGEDS LAALSSVPCE APISDPFAPE PSPPTTTTEP ASASASTTTA VTAVTTEVDL FGDAFAASPG EAPAASEGAT APATPAPVAA ALDACSGNDP FAPSEGSAEA APELDLFAMK PPETSAPVVT PTASTAPPVP ATAPSPAPTA VAATAATTTA AAAATTTATT SAAAATTAAA PPALDIFGDL FDSAPEVAAA PKPDAAPSID LFGTDAFSSP PRGASPVPES SLTADLLSVD AFAAPSPAST ASPAKAESSG VIDLFGDAFG SGASETQPAP QAVSSSSASA DLLAGFGGSF MAPSTTPVTP AQNNLLQPSF EAAFGTTPST SSSSSFDPSV FDGLGDLLMP TMAPSGQPAP VSMVPPSPAM AASKGLGSDL DSSLASLVGN LGISGTTSKK GDLQWNAGEK KLTGGANWQP KVTPATWSAG VPPQGTVPPT SSVPPGAGAP SVGQPGAGFG MPPSGTGMTM MSQQPVMFAQ PMMRPPFGAA AVPGTQLSPS PTPATQSPKK PPAKDPLADL NIKDFL |
-Macromolecule #2: auxilin
Macromolecule | Name: auxilin / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Bos taurus (cattle) |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: PSGPTSTQST PRRSATSTSA SPTLRVGEGA TFDPFGAPSK PSGQDLLGSF LNTASASSDP FLQPTRSPSP TVHASSTPAV NIQPDVSGAW DWHTKPGGFG MGSKSAATSP TGSSHGTPTH QNKPQTLDPF ADLGTLGGSS FASKPSTPTG LGGGFPPLSS PQKASPQPMG ...String: PSGPTSTQST PRRSATSTSA SPTLRVGEGA TFDPFGAPSK PSGQDLLGSF LNTASASSDP FLQPTRSPSP TVHASSTPAV NIQPDVSGAW DWHTKPGGFG MGSKSAATSP TGSSHGTPTH QNKPQTLDPF ADLGTLGGSS FASKPSTPTG LGGGFPPLSS PQKASPQPMG GGWQQGGGYN WQQTQSKPQS SMPHSSPQNR PNYNVSFSSM PGGQNERGKA AANLEGKQKA ADFEDLLSGQ GFNAHKDKKG PRTIAEMRKE EMAKEMDPEK LKILEWIEGK ERNIRALLST MHTVLWAGET KWKPVGMADL VTPEQVKKVY RKAVLVVHPD KATGQPYEQY AKMIFMELND AWSEFENQGQ KPLY |
-Macromolecule #3: clathrin light chain A1
Macromolecule | Name: clathrin light chain A1 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: Rattus norvegicus (Norway rat) |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MAELDPFGAP AGAPGGPALG NGVAGAGEED PAAAFLAQQE SEIAGIENDE AFAILDGGAP GPQAHGEPPG GPDAVDGVMN GEYYQESNGP TDSYAAISEV DRLQSEPESI RKWREEQTER LEALDANSRK QEAEWKEKAV KELEEWYARQ DEQLQKTKAS NRVADEAFYK ...String: MAELDPFGAP AGAPGGPALG NGVAGAGEED PAAAFLAQQE SEIAGIENDE AFAILDGGAP GPQAHGEPPG GPDAVDGVMN GEYYQESNGP TDSYAAISEV DRLQSEPESI RKWREEQTER LEALDANSRK QEAEWKEKAV KELEEWYARQ DEQLQKTKAS NRVADEAFYK QPFADVIGYV TNINHPCYSL EQAAEEAFVN DIDESSPGTE WERVARLCDF NPKSSKQAKD VSRMRSVLIS LKQAPLVH |
-Macromolecule #4: clathrin heavy chain 1
Macromolecule | Name: clathrin heavy chain 1 / type: protein_or_peptide / ID: 4 / Details: His tag at N-terminus / Enantiomer: LEVO |
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Source (natural) | Organism: Rattus norvegicus (Norway rat) |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MGSSHHHHHH SSGLVPRGSH MASMTGGQQM GRGSEFELRR QAWMAQILPI RFQEHLQLQN LGINPANIGF STLTMESDKF ICIREKVGEQ AQVVIIDMND PSNPIRRPIS ADSAIMNPAS KVIALKAGKT LQIFNIEMKS KMKAHTMTDD VTFWKWISLN TVALVTDNAV ...String: MGSSHHHHHH SSGLVPRGSH MASMTGGQQM GRGSEFELRR QAWMAQILPI RFQEHLQLQN LGINPANIGF STLTMESDKF ICIREKVGEQ AQVVIIDMND PSNPIRRPIS ADSAIMNPAS KVIALKAGKT LQIFNIEMKS KMKAHTMTDD VTFWKWISLN TVALVTDNAV YHWSMEGESQ PVKMFDRHSS LAGCQIINYR TDAKQKWLLL TGISAQQNRV VGAMQLYSVD RKVSQPIEGH AASFAQFKME GNAEESTLFC FAVRGQAGGK LHIIEVGTPP TGNQPFPKKA VDVFFPPEAQ NDFPVAMQIS EKHDVVFLIT KYGYIHLYDL ETGTCIYMNR ISGETIFVTA PHEATAGIIG VNRKGQVLSV CVEEENIIPY ITNVLQNPDL ALRMAVRNNL AGAEELFARK FNALFAQGNY SEAAKVAANA PKGILRTPDT IRRFQSVPAQ PGQTSPLLQY FGILLDQGQL NKYESLELCR PVLQQGRKQL LEKWLKEDKL ECSEELGDLV KSVDPTLALS VYLRANVPNK VIQCFAETGQ VQKIVLYAKK VGYTPDWIFL LRNVMRISPD QGQQFAQMLV QDEEPLADIT QIVDVFMEYN LIQQCTAFLL DALKNNRPSE GPLQTRLLEM NLMHAPQVAD AILGNQMFTH YDRAHIAQLC EKAGLLQRAL EHFTDLYDIK RAVVHTHLLN PEWLVNYFGS LSVEDSLECL RAMLSANIRQ NLQIWVQVAS KYHEQLSTQS LIELFESFKS FEGLFYFLGS IVNFSQDPDV HFKYIQAACK TGQIKEVERI CRESNCYDPE RVKNFLKEAK LTDQLPLIIV CDRFDFVHDL VLYLYRNSLQ KYIEIYVQKV NPSRLPVVIG GLLDVDCSED VIKNLILVVR GQFSTDELVA EVEKRNRLKL LLPWLEARIH EGCEEPATHN ALAKIYIDSN NNPERFLREN PYYDSRVVGK YCEKRDPHLA CVAYERGQCD LELINVCNEN SLFKSLSRYL VRRKDPELWG SVLLESNPYR RPLIDQVVQT ALSETQDPEE VSVTVKAFMT ADLPNELIEL LEKIVLDNSV FSEHRNLQNL LILTAIKADR TRVMEYINRL DNYDAPDIAN IAISNELFEE AFAIFRKFDV NTSAVQVLIE HIGNLDRAYE FAERCNEPAV WSQLAKAQLQ KGMVKEAIDS YIKADDPSSY MEVVQAANTS GNWEELVKYL QMARKKARES YVETELIFAL AKTNRLAELE EFINGPNNAH IQQVGDRCYD EKMYDAAKLL YNNVSNFGRL ASTLVHLGEY QAAVDGARKA NSTRTWKEVC FACVDGKEFR LAQMCGLHIV VHADELEELI NYYQDRGYFE ELITMLEAAL GLERAHMGMF TELAILYSKF KPQKMREHLE LFWSRVNIPK VLRAAEQAHL WAELVFLYDK YEEYDNAIIT MMNHPTDAWK EGQFKDIITK VANVELYYKA IQFYLEFKPL LLNDLLMVLS PRLAHTRAVN YFSKVKQLPL VKPYLRSVQN HNNKSVNESL NNLFITEEDY QALRTSIDAY DNFDNISLAQ RLEKHELIEF RRIAAYLFKG NNRWKQSVEL CKKDSLYKDA MQYASESKDT ELAEELLQWF LQEEKRECFG ACLFTCYDLL RPDVVLETAW RHNIMDFAMP YFIQVMKEYL TKVDKLDASE SLRKEEEQAT ETQPIVYGQP QLMLTAGPSV AVPPQAPFGY GYTAPPYGQP QPGFGYSM |
-Macromolecule #5: Hsc70deltaC
Macromolecule | Name: Hsc70deltaC / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO |
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Source (natural) | Organism: Bos taurus (cattle) |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
Sequence | String: MSKGPAVGID LGTTYSCVGV FQHGKVEIIA NDQGNRTTPS YVAFTDTERL IGDAAKNQVA MNPTNTVFDA KRLIGRRFDD AVVQSDMKHW PFMVVNDAGR PKVQVEYKGE TKSFYPEEVS SMVLTKMKEI AEAYLGKTVT NAVVTVPAYF NDSQRQATKD AGTIAGLNVL ...String: MSKGPAVGID LGTTYSCVGV FQHGKVEIIA NDQGNRTTPS YVAFTDTERL IGDAAKNQVA MNPTNTVFDA KRLIGRRFDD AVVQSDMKHW PFMVVNDAGR PKVQVEYKGE TKSFYPEEVS SMVLTKMKEI AEAYLGKTVT NAVVTVPAYF NDSQRQATKD AGTIAGLNVL RIINEPTAAA IAYGLDKKVG AERNVLIFDL GGGTFDVSIL TIEDGIFEVK STAGDTHLGG EDFDNRMVNH FIAEFKRKHK KDISENKRAV RRLRTACERA KRTLSSSTQA SIEIDSLYEG IDFYTSITRA RFEELNADLF RGTLDPVEKA LRDAKLDKSQ IHDIVLVGGS TRIPKIQKLL QDFFNGKELN KSINPDEAVA YGAAVQAAIL SGDKSENVQD LLLLDVTPLS LGIETAGGVM TVLIKRNTTI PTKQTQTFTT YSDNQPGVLI QVYEGERAMT KDNNLLGKFE LTGIPPAPRG VPQIEVTFDI DANGILNVSA VDKSTGKENK ITITNDKGRL SKEDIERMVQ EAEKYKAEDE KQRDKVSSKN SLESYAFNMK ATVE |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 6 Details: 2 mM MgCl2 25 mM KCl 10 mM (NH4)2SO4 2 mM DTT 20 mM MES pH 6.0 |
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Grid | Model: QUANTIFOIL / Material: COPPER/RHODIUM / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 400.0 nm / Pretreatment - Type: GLOW DISCHARGE |
Vitrification | Cryogen name: ETHANE / Chamber temperature: 90 K / Instrument: LEICA EM CPC |
-Electron microscopy
Microscope | FEI TECNAI F20 |
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Temperature | Min: 90.0 K / Max: 98.0 K |
Image recording | Film or detector model: FEI EAGLE (4k x 4k) / Digitization - Dimensions - Width: 4000 pixel / Digitization - Dimensions - Height: 4000 pixel / Digitization - Sampling interval: 14.6 µm / Number grids imaged: 1 / Number real images: 475 / Average exposure time: 1.5 sec. / Average electron dose: 9.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 41000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.26 mm / Nominal defocus max: 8.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 30000 |
Sample stage | Specimen holder model: GATAN LIQUID NITROGEN / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company |