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- EMDB-2410: Hsc70-induced Changes in Clathrin-Auxilin Cage Structure Suggest ... -

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Entry
Database: EMDB / ID: EMD-2410
TitleHsc70-induced Changes in Clathrin-Auxilin Cage Structure Suggest a Role for Clathrin Light Chains in Cage Disassembly
Map dataClathrin-auxilin-hsc70 complex
Sample
  • Sample: Clathrin, auxilin (residues 401-910) and Hsc70
  • Protein or peptide: Hsc70HSPA8
  • Protein or peptide: auxilin401-910
KeywordsEndocytosis / coated vesicles / clathrin / hsc70 / auxilin
Biological speciesRattus (rat) / Bos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 34.0 Å
AuthorsYoung A / Stoilova-McPhie S / Rothnie A / Vallis Y / Harvey-Smith P / Ranson N / Kent H / Brodsky FM / Pearse BM / Roseman A / Smith CJ
CitationJournal: Traffic / Year: 2013
Title: Hsc70-induced changes in clathrin-auxilin cage structure suggest a role for clathrin light chains in cage disassembly.
Authors: Anna Young / Svetla Stoilova-McPhie / Alice Rothnie / Yvonne Vallis / Phillip Harvey-Smith / Neil Ranson / Helen Kent / Frances M Brodsky / Barbara M F Pearse / Alan Roseman / Corinne J Smith /
Abstract: The molecular chaperone, Hsc70, together with its co-factor, auxilin, facilitates the ATP-dependent removal of clathrin during clathrin-mediated endocytosis in cells. We have used cryo-electron ...The molecular chaperone, Hsc70, together with its co-factor, auxilin, facilitates the ATP-dependent removal of clathrin during clathrin-mediated endocytosis in cells. We have used cryo-electron microscopy to determine the 3D structure of a complex of clathrin, auxilin(401-910) and Hsc70 at pH 6 in the presence of ATP, frozen within 20 seconds of adding Hsc70 in order to visualize events that follow the binding of Hsc70 to clathrin and auxilin before clathrin disassembly. In this map, we observe density beneath the vertex of the cage that we attribute to bound Hsc70. This density emerges asymmetrically from the clathrin vertex, suggesting preferential binding by Hsc70 for one of the three possible sites at the vertex. Statistical comparison with a map of whole auxilin and clathrin previously published by us reveals the location of statistically significant differences which implicate involvement of clathrin light chains in structural rearrangements which occur after Hsc70 is recruited. Clathrin disassembly assays using light scattering suggest that loss of clathrin light chains reduces the efficiency with which auxilin facilitates this reaction. These data support a regulatory role for clathrin light chains in clathrin disassembly in addition to their established role in regulating clathrin assembly.
History
DepositionJul 2, 2013-
Header (metadata) releaseJul 10, 2013-
Map releaseJul 10, 2013-
UpdateAug 14, 2013-
Current statusAug 14, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 29
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 29
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

image.png

Downloads & links

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Map

FileDownload / File: emd_2410.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationClathrin-auxilin-hsc70 complex
Voxel sizeX=Y=Z: 6.4 Å
Density
Contour LevelBy AUTHOR: 29.0 / Movie #1: 29
Minimum - Maximum-49.826423650000002 - 111.215446470000003
Average (Standard dev.)0.0 (±5.78422499)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-128-128-128
Dimensions256256256
Spacing256256256
CellA=B=C: 1638.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z6.46.46.4
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z1638.4001638.4001638.400
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS-128-128-128
NC/NR/NS256256256
D min/max/mean-49.826111.2150.000

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Supplemental data

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Sample components

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Entire : Clathrin, auxilin (residues 401-910) and Hsc70

EntireName: Clathrin, auxilin (residues 401-910) and Hsc70
Components
  • Sample: Clathrin, auxilin (residues 401-910) and Hsc70
  • Protein or peptide: Hsc70HSPA8
  • Protein or peptide: auxilin401-910

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Supramolecule #1000: Clathrin, auxilin (residues 401-910) and Hsc70

SupramoleculeName: Clathrin, auxilin (residues 401-910) and Hsc70 / type: sample / ID: 1000 / Oligomeric state: Not experimentally determined / Number unique components: 3

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Macromolecule #1: Hsc70

MacromoleculeName: Hsc70 / type: protein_or_peptide / ID: 1 / Recombinant expression: Yes
Source (natural)Organism: Rattus (rat) / synonym: Rat

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Macromolecule #2: auxilin401-910

MacromoleculeName: auxilin401-910 / type: protein_or_peptide / ID: 2 / Recombinant expression: Yes
Source (natural)Organism: Bos taurus (cattle) / synonym: Cattle

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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: 20mM MES, 2mM magnesium acetate, 25mM KCl, 10mM (NH4)2SO4, 1.8mM Hepes, 18mM NaCl, 1mM DTT
GridDetails: Quantifoil holey carbon grids, glow discharged in amylamine atmosphere
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
Timed resolved state: Vitrified rapidly following addition of Hsc70-ATP

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Electron microscopy

MicroscopeJEOL 2011
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 40000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
DateJan 1, 2004
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON COOLSCAN

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Image processing

CTF correctionDetails: Whole micrographs
Final reconstructionApplied symmetry - Point group: D6 (2x6 fold dihedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 34.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: MRC, SPIDER, FREALIGN
Details: The EM map has been sharpened to match a Fourier amplitude profile derived from a model of the hexagonal barrel (the biological unit) created from the crystal structure atomic model pdb ...Details: The EM map has been sharpened to match a Fourier amplitude profile derived from a model of the hexagonal barrel (the biological unit) created from the crystal structure atomic model pdb id1XI5. Fourier filtered to 30 Angstroms.
Number images used: 1051

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