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- EMDB-4036: Cryo-EM reconstruction of clathrin D6 cages + Hsc70 Delta C -

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Basic information

Entry
Database: EMDB / ID: EMD-4036
TitleCryo-EM reconstruction of clathrin D6 cages + Hsc70 Delta C
Map dataNone
Sample
  • Complex: Cryo-EM reconstruction of clathrin D6 cages + Hsc70 Delta C
    • Protein or peptide: AP180
    • Protein or peptide: auxilin
    • Protein or peptide: clathrin light chain A1
    • Protein or peptide: clathrin heavy chain 1
    • Protein or peptide: Hsc70deltaC
Biological speciesMus musculus (house mouse) / Bos taurus (cattle) / Rattus norvegicus (Norway rat)
Methodsingle particle reconstruction / cryo EM / Resolution: 28.0 Å
AuthorsSousa R / Liao H-S / Cuellar J / Valpuesta JM / Jin AJ / Lafer EM
CitationJournal: 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
DepositionJun 21, 2016-
Header (metadata) releaseJul 27, 2016-
Map releaseJul 27, 2016-
UpdateJul 12, 2017-
Current statusJul 12, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.73
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 1.73
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4036.png

Downloads & links

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Map

FileDownload / File: emd_4036.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Voxel sizeX=Y=Z: 3.65 Å
Density
Contour LevelBy AUTHOR: 1.73 / Movie #1: 1.73
Minimum - Maximum0. - 10.145052
Average (Standard dev.)0.228938 (±0.8608905)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 1168.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.653.653.65
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z1168.0001168.0001168.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean0.00010.1450.229

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Supplemental data

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Sample components

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Entire : Cryo-EM reconstruction of clathrin D6 cages + Hsc70 Delta C

EntireName: Cryo-EM reconstruction of clathrin D6 cages + Hsc70 Delta C
Components
  • Complex: Cryo-EM reconstruction of clathrin D6 cages + Hsc70 Delta C
    • Protein or peptide: AP180
    • Protein or peptide: auxilin
    • Protein or peptide: clathrin light chain A1
    • Protein or peptide: clathrin heavy chain 1
    • Protein or peptide: Hsc70deltaC

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Supramolecule #1: Cryo-EM reconstruction of clathrin D6 cages + Hsc70 Delta C

SupramoleculeName: Cryo-EM reconstruction of clathrin D6 cages + Hsc70 Delta C
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 31.476968 MDa

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Macromolecule #1: AP180

MacromoleculeName: AP180 / type: protein_or_peptide / ID: 1 / Details: GST tag at N-terminus / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: 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

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Macromolecule #2: auxilin

MacromoleculeName: auxilin / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (cattle)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: 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

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Macromolecule #3: clathrin light chain A1

MacromoleculeName: clathrin light chain A1 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: 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

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Macromolecule #4: clathrin heavy chain 1

MacromoleculeName: clathrin heavy chain 1 / type: protein_or_peptide / ID: 4 / Details: His tag at N-terminus / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: 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

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Macromolecule #5: Hsc70deltaC

MacromoleculeName: Hsc70deltaC / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (cattle)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: 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

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 6
Details: 2 mM MgCl2 25 mM KCl 10 mM (NH4)2SO4 2 mM DTT 20 mM MES pH 6.0
GridModel: 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
VitrificationCryogen name: ETHANE / Chamber temperature: 90 K / Instrument: LEICA EM CPC

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 41000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.26 mm / Nominal defocus max: 8.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 30000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN / Cooling holder cryogen: NITROGEN
TemperatureMin: 90.0 K / Max: 98.0 K
Image recordingFilm 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
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 5609
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: EMDB MAP
EMDB ID:

3442

Initial angle assignmentType: PROJECTION MATCHING / Software - Name: Xmipp (ver. 3.0)
Final 3D classificationNumber classes: 3 / Avg.num./class: 1800 / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: Xmipp (ver. 3.0)
Final reconstructionApplied symmetry - Point group: D6 (2x6 fold dihedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 28.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Xmipp (ver. 3.0) / Number images used: 2300

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