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- EMDB-3442: Cryo-EM reconstructions of clathrin D6 cages -

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Basic information

Entry
Database: EMDB / ID: EMD-3442
TitleCryo-EM reconstructions of clathrin D6 cages
Map dataCryo-EM reconstructions of clathrin D6 cages
Sample
  • Sample: Clathrin D6 Coat
  • Protein or peptide: Mouse AP180 residues 305-901
  • Protein or peptide: Bovine Auxilin residues 547-910
  • Protein or peptide: Rat Clathrin Light Chain A1: residues 1-248
  • Protein or peptide: Rat Clathrin Heavy Chain 1: residues 1-1675
KeywordsClatrhin coats / Hsc70 / auxilin / ATP / disassembly / self-association / entropic pulling
Function / homology
Function and homology information


negative regulation of phospholipase D activity / presynaptic endosome / positive regulation of clathrin coat assembly / postsynaptic endocytic zone cytoplasmic component / extrinsic component of endosome membrane / RHOU GTPase cycle / RHOV GTPase cycle / regulation of clathrin coat assembly / presynaptic endocytic zone / postsynaptic endocytic zone ...negative regulation of phospholipase D activity / presynaptic endosome / positive regulation of clathrin coat assembly / postsynaptic endocytic zone cytoplasmic component / extrinsic component of endosome membrane / RHOU GTPase cycle / RHOV GTPase cycle / regulation of clathrin coat assembly / presynaptic endocytic zone / postsynaptic endocytic zone / synaptic vesicle budding from presynaptic endocytic zone membrane / presynaptic endocytic zone membrane / Myb complex / regulation of clathrin-dependent endocytosis / 1-phosphatidylinositol binding / clathrin coat of trans-Golgi network vesicle / clathrin vesicle coat / Gap junction degradation / Formation of annular gap junctions / regulation of terminal button organization / negative regulation of hyaluronan biosynthetic process / positive regulation of synaptic vesicle clustering / clathrin light chain binding / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / synaptic vesicle recycling / LDL clearance / VLDLR internalisation and degradation / Retrograde neurotrophin signalling / clathrin complex / regulation of protein transport / clathrin coat / WNT5A-dependent internalization of FZD4 / Lysosome Vesicle Biogenesis / synaptic vesicle uncoating / extrinsic component of presynaptic endocytic zone membrane / negative regulation of phospholipase C activity / Golgi Associated Vesicle Biogenesis / amyloid-beta clearance by transcytosis / transferrin transport / MHC class II antigen presentation / extrinsic component of synaptic vesicle membrane / clathrin heavy chain binding / clathrin coat of coated pit / AP-2 adaptor complex / mitotic spindle microtubule / photoreceptor ribbon synapse / clathrin coat disassembly / Recycling pathway of L1 / clathrin coat assembly / inositol hexakisphosphate binding / Cargo recognition for clathrin-mediated endocytosis / Cargo recognition for clathrin-mediated endocytosis / clathrin-coated endocytic vesicle / positive regulation of synaptic vesicle endocytosis / Clathrin-mediated endocytosis / clathrin adaptor activity / Clathrin-mediated endocytosis / vesicle budding from membrane / Hydrolases; Acting on ester bonds; Phosphoric-monoester hydrolases / membrane coat / neurotransmitter secretion / clathrin-dependent endocytosis / protein serine/threonine kinase binding / retrograde transport, endosome to Golgi / negative regulation of protein localization to plasma membrane / parallel fiber to Purkinje cell synapse / low-density lipoprotein particle receptor binding / clathrin-coated vesicle / regulation of synaptic vesicle endocytosis / ankyrin binding / establishment or maintenance of cell polarity / clathrin binding / positive regulation of axonogenesis / ubiquitin-specific protease binding / Golgi organization / phospholipase binding / synaptic vesicle endocytosis / intracellular transport / regulation of endocytosis / mitotic spindle assembly / neuron development / axon terminus / dephosphorylation / presynaptic active zone membrane / regulation of mitotic spindle organization / clathrin-coated pit / phosphatidylinositol-4,5-bisphosphate binding / T-tubule / heat shock protein binding / receptor-mediated endocytosis / SH2 domain binding / axonogenesis / SNARE binding / protein tyrosine phosphatase activity / peptide binding / clathrin-coated endocytic vesicle membrane / intracellular protein transport / sarcolemma / Schaffer collateral - CA1 synapse / terminal bouton
Similarity search - Function
Clathrin coat assembly protein AP180 / ANTH domain superfamily / Clathrin light chain / Clathrin light chain signature 1. / Clathrin light chain signature 2. / Clathrin light chain / AP180 N-terminal homology (ANTH) domain / ANTH domain / Clathrin-H-link / Epsin N-terminal homology (ENTH) domain ...Clathrin coat assembly protein AP180 / ANTH domain superfamily / Clathrin light chain / Clathrin light chain signature 1. / Clathrin light chain signature 2. / Clathrin light chain / AP180 N-terminal homology (ANTH) domain / ANTH domain / Clathrin-H-link / Epsin N-terminal homology (ENTH) domain / Clathrin, heavy chain, linker, core motif / Clathrin heavy chain, N-terminal / Clathrin, heavy chain / Clathrin, heavy chain, propeller repeat / Clathrin propeller repeat / Clathrin, heavy-chain linker / ENTH domain profile. / Region in Clathrin and VPS / Tensin phosphatase, C2 domain / Tensin-type phosphatase domain / C2 domain of PTEN tumour-suppressor protein / Phosphatase tensin-type domain profile. / C2 tensin-type domain profile. / Clathrin heavy chain repeat homology / C2 domain of PTEN tumour-suppressor protein / ENTH domain / Clathrin, heavy chain/VPS, 7-fold repeat / Clathrin heavy-chain (CHCR) repeat profile. / ENTH/VHS / DnaJ molecular chaperone homology domain / dnaJ domain profile. / Chaperone J-domain superfamily / DnaJ domain / Tyrosine-specific protein phosphatases domain / Tyrosine specific protein phosphatases domain profile. / C2 domain superfamily / Protein-tyrosine phosphatase-like / Tetratricopeptide-like helical domain superfamily / Armadillo-type fold
Similarity search - Domain/homology
Clathrin light chain A / Clathrin heavy chain 1 / Auxilin / Clathrin coat assembly protein AP180
Similarity search - Component
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 20, 2016-
Header (metadata) releaseJul 27, 2016-
Map releaseJul 27, 2016-
UpdateAug 3, 2016-
Current statusAug 3, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.5
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 2.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

3442_imageCl...

Downloads & links

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Map

FileDownload / File: emd_3442.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM reconstructions of clathrin D6 cages
Voxel sizeX=Y=Z: 3.65 Å
Density
Contour LevelBy AUTHOR: 2.5 / Movie #1: 2.5
Minimum - Maximum-1.43189907 - 13.58245754
Average (Standard dev.)0.0 (±1.0)
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/mean-1.43213.582-0.000

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Supplemental data

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Sample components

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Entire : Clathrin D6 Coat

EntireName: Clathrin D6 Coat
Components
  • Sample: Clathrin D6 Coat
  • Protein or peptide: Mouse AP180 residues 305-901
  • Protein or peptide: Bovine Auxilin residues 547-910
  • Protein or peptide: Rat Clathrin Light Chain A1: residues 1-248
  • Protein or peptide: Rat Clathrin Heavy Chain 1: residues 1-1675

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Supramolecule #1000: Clathrin D6 Coat

SupramoleculeName: Clathrin D6 Coat / type: sample / ID: 1000 / Oligomeric state: 36 triskelia / Number unique components: 4
Molecular weightTheoretical: 31.5 MDa

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Macromolecule #1: Mouse AP180 residues 305-901

MacromoleculeName: Mouse AP180 residues 305-901 / type: protein_or_peptide / ID: 1 / Details: GST tag at N-terminus. / Number of copies: 36 / Recombinant expression: Yes
Source (natural)Organism: Mus musculus (house mouse) / synonym: Mouse
Molecular weightExperimental: 84.268 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21
SequenceUniProtKB: Clathrin coat assembly protein AP180

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Macromolecule #2: Bovine Auxilin residues 547-910

MacromoleculeName: Bovine Auxilin residues 547-910 / type: protein_or_peptide / ID: 2 / Number of copies: 108 / Recombinant expression: Yes
Source (natural)Organism: Bos taurus (cattle) / synonym: Cow
Molecular weightExperimental: 38.935 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21
SequenceUniProtKB: Auxilin

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Macromolecule #3: Rat Clathrin Light Chain A1: residues 1-248

MacromoleculeName: Rat Clathrin Light Chain A1: residues 1-248 / type: protein_or_peptide / ID: 3 / Name.synonym: CLC, CLCA, CLCA1 / Number of copies: 108 / Recombinant expression: Yes
Source (natural)Organism: Rattus norvegicus (Norway rat) / synonym: Rat
Molecular weightExperimental: 26.979 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21
SequenceUniProtKB: Clathrin light chain A

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Macromolecule #4: Rat Clathrin Heavy Chain 1: residues 1-1675

MacromoleculeName: Rat Clathrin Heavy Chain 1: residues 1-1675 / type: protein_or_peptide / ID: 4 / Name.synonym: CHC, CHC1 / Details: His tag at the N-terminus / Number of copies: 108 / Recombinant expression: Yes
Source (natural)Organism: Rattus norvegicus (Norway rat) / synonym: Rat
Molecular weightExperimental: 196.358 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21
SequenceUniProtKB: Clathrin heavy chain 1

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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: 20 mM MES, 2 mM MgCl2, 25 mM KCl, 10 mM (NH4)2SO4 and 2 mM DTT
GridDetails: Cu/Rh 300 mesh Quantifoil R 1.2/1.3 um grids with thin carbon support
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 98 K / Instrument: LEICA EM CPC / Method: Blot for 1 second before plunging

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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
TemperatureMin: 90 K / Max: 98 K / Average: 94 K
DateJun 2, 2015
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Digitization - Sampling interval: 14.6 µm / Number real images: 450 / Average electron dose: 22 e/Å2 / Bits/pixel: 8
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: cctffind3
Final two d classificationNumber classes: 10
Final reconstructionApplied symmetry - Point group: D6 (2x6 fold dihedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 28.0 Å / Resolution method: OTHER / Software - Name: XMIPP, Relion / Number images used: 3468
DetailsManual picking

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