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- EMDB-2411: Hsc70-induced Changes in Clathrin-Auxilin Cage Structure Suggest ... -

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Basic information

Entry
Database: EMDB / ID: EMD-2411
TitleHsc70-induced Changes in Clathrin-Auxilin Cage Structure Suggest a Role for Clathrin Light Chains in Cage Disassembly
Map dataMap of Clathrin-Auxilin complex. 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.
Sample
  • Sample: Clathrin bound to auxilin
  • Protein or peptide: Clathrin
  • Protein or peptide: Auxilin
KeywordsEndocytosis / coated vesicles / clathrin / auxilin
Biological speciesSus scrofa (pig)
Methodsingle particle reconstruction / cryo EM / Resolution: 31.0 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: J Mol Biol / Year: 2004
Title: Location of auxilin within a clathrin cage.
Authors: Corinne J Smith / Timothy R Dafforn / Helen Kent / Catherine A Sims / Kavita Khubchandani-Aswani / Lin Zhang / Helen R Saibil / Barbara M F Pearse /
Abstract: The Dna J homologue, auxilin, acts as a co-chaperone for Hsc70 in the uncoating of clathrin-coated vesicles during endocytosis. Biochemical studies have aided understanding of the uncoating mechanism ...The Dna J homologue, auxilin, acts as a co-chaperone for Hsc70 in the uncoating of clathrin-coated vesicles during endocytosis. Biochemical studies have aided understanding of the uncoating mechanism but until now there was no structural information on how auxilin interacts with the clathrin cage. Here we have determined the three-dimensional structure of a complex of auxilin with clathrin cages by cryo-electron microscopy and single particle analysis. We show that auxilin forms a discrete shell of density on the inside of the clathrin cage. Peptide competition assays confirm that a candidate clathrin box motif in auxilin, LLGLE, can bind to a clathrin construct containing the beta-propeller domain and also displace the well-characterised LLNLD clathrin box motif derived from the beta-adaptin hinge region. The means by which auxilin could both aid clathrin coat assembly and displace clathrin from AP2 during uncoating is discussed.
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_2411.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of Clathrin-Auxilin complex. 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.
Voxel sizeX=Y=Z: 6.4 Å
Density
Contour LevelBy AUTHOR: 29.0 / Movie #1: 29
Minimum - Maximum-57.857616419999999 - 109.673622129999998
Average (Standard dev.)0.0 (±5.80865669)
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-57.858109.674-0.000

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Supplemental data

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Sample components

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Entire : Clathrin bound to auxilin

EntireName: Clathrin bound to auxilin
Components
  • Sample: Clathrin bound to auxilin
  • Protein or peptide: Clathrin
  • Protein or peptide: Auxilin

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Supramolecule #1000: Clathrin bound to auxilin

SupramoleculeName: Clathrin bound to auxilin / type: sample / ID: 1000 / Number unique components: 2

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Macromolecule #1: Clathrin

MacromoleculeName: Clathrin / type: protein_or_peptide / ID: 1 / Recombinant expression: No
Source (natural)Organism: Sus scrofa (pig)

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Macromolecule #2: Auxilin

MacromoleculeName: Auxilin / type: protein_or_peptide / ID: 2 / Recombinant expression: No
Source (natural)Organism: Sus scrofa (pig)

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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 (pH 6.0), 2 mM magnesium acetate, 25 mM KCl, 10 mM (NH4)2SO4, 1 mM DTT
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER

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Electron microscopy #1

MicroscopeFEI/PHILIPS CM200FEG
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Microscopy ID1
DateFeb 2, 2000
Image recordingDigitization - Scanner: ZEISS SCAI

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Electron microscopy #2

MicroscopeFEI/PHILIPS CM200FEG
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Microscopy ID2
DateFeb 23, 2000
Image recordingDigitization - Scanner: ZEISS SCAI

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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: 31.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: MRC, SPIDER, FREALIGN
Details: 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: 1745

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