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- EMDB-5118: Clathrin D6 coat with Hsc70 and Auxilin -

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Basic information

Entry
Database: EMDB / ID: EMD-5118
TitleClathrin D6 coat with Hsc70 and Auxilin
Map dataThis is a volume of a D6 clathrin coat bound with Hsc70(1-554) and Auxilin(547-910)
Sample
  • Sample: Clathrin coats bound with Hsc70(1-554) and Auxilin(547-910)
  • Protein or peptide: uncoating intermediate of clathrin coat
Keywordsclathrin / D6 coat / Hsc70 / Auxilin / uncoating
Biological speciesBos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 15.2 Å
AuthorsXing Y / Boecking T / Wolf M / Kirchhausen T / Harrison SC
CitationJournal: EMBO J / Year: 2010
Title: Structure of clathrin coat with bound Hsc70 and auxilin: mechanism of Hsc70-facilitated disassembly.
Authors: Yi Xing / Till Böcking / Matthias Wolf / Nikolaus Grigorieff / Tomas Kirchhausen / Stephen C Harrison /
Abstract: The chaperone Hsc70 drives the clathrin assembly-disassembly cycle forward by stimulating dissociation of a clathrin lattice. A J-domain containing co-chaperone, auxilin, associates with a freshly ...The chaperone Hsc70 drives the clathrin assembly-disassembly cycle forward by stimulating dissociation of a clathrin lattice. A J-domain containing co-chaperone, auxilin, associates with a freshly budded clathrin-coated vesicle, or with an in vitro assembled clathrin coat, and recruits Hsc70 to its specific heavy-chain-binding site. We have determined by electron cryomicroscopy (cryoEM), at about 11 A resolution, the structure of a clathrin coat (in the D6-barrel form) with specifically bound Hsc70 and auxilin. The Hsc70 binds a previously analysed site near the C-terminus of the heavy chain, with a stoichiometry of about one per three-fold vertex. Its binding is accompanied by a distortion of the clathrin lattice, detected by a change in the axial ratio of the D6 barrel. We propose that when Hsc70, recruited to a position close to its target by the auxilin J-domain, splits ATP, it clamps firmly onto its heavy-chain site and locks in place a transient fluctuation. Accumulation of the local strain thus imposed at multiple vertices can then lead to disassembly.
History
DepositionJun 4, 2009-
Header (metadata) releaseFeb 11, 2010-
Map releaseFeb 11, 2010-
UpdateFeb 11, 2010-
Current statusFeb 11, 2010Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5118_1.tif

Downloads & links

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Map

FileDownload / File: emd_5118.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a volume of a D6 clathrin coat bound with Hsc70(1-554) and Auxilin(547-910)
Voxel sizeX=Y=Z: 4.2 Å
Density
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-0.919363 - 2.08404
Average (Standard dev.)-0.0175207 (±0.259777)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-128-128-128
Dimensions256256256
Spacing256256256
CellA=B=C: 1075.2 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.24.24.2
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z1075.2001075.2001075.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-34-26-72
NX/NY/NZ6953145
MAP C/R/S123
start NC/NR/NS-128-128-128
NC/NR/NS256256256
D min/max/mean-0.9192.084-0.018

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Supplemental data

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Sample components

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Entire : Clathrin coats bound with Hsc70(1-554) and Auxilin(547-910)

EntireName: Clathrin coats bound with Hsc70(1-554) and Auxilin(547-910)
Components
  • Sample: Clathrin coats bound with Hsc70(1-554) and Auxilin(547-910)
  • Protein or peptide: uncoating intermediate of clathrin coat

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Supramolecule #1000: Clathrin coats bound with Hsc70(1-554) and Auxilin(547-910)

SupramoleculeName: Clathrin coats bound with Hsc70(1-554) and Auxilin(547-910)
type: sample / ID: 1000
Details: clathrin coats assembled from clathrin and AP-2 with excess of Hsc70(1-554) and Auxilin(547-910) added
Number unique components: 9

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Macromolecule #1: uncoating intermediate of clathrin coat

MacromoleculeName: uncoating intermediate of clathrin coat / type: protein_or_peptide / ID: 1
Name.synonym: clathrin coat in complex with Hsc70 and auxilin
Number of copies: 108 / Oligomeric state: D6 assemble / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (cattle) / synonym: cattle / Tissue: Brain

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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

Concentration0.25 mg/mL
BufferpH: 6
Details: 20mM MES, pH6.0, 2mM MgCl2, 25mM KCl, 10mM (NH4)2SO4, and 2mM DTT
GridDetails: holey carbon grid
VitrificationCryogen name: ETHANE / Chamber humidity: 60 % / Chamber temperature: 93 K / Instrument: OTHER / Details: Vitrification instrument: FEI Vitrobot
Timed resolved state: Vitrified 30 msec after spraying with effector
Method: Blot for 2 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 51159 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder: side entry liquid nitrogen-cooled cryo specimen holder.
Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 93 K
DateJan 1, 2007
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 900 / Average electron dose: 20 e/Å2 / Od range: 1.4 / Bits/pixel: 8
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: CTF correction of each particle.
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 15.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Frealign
Details: a raw map without B factor sharpening and n.c.s averaging
Number images used: 1500

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Atomic model buiding 1

Initial model(PDB ID:

1n4c

,

1bpo

,

1b89

)
DetailsProtocol: Rigid Body. Various segments of clathrin heavy chain ere separately fitted by manual docking using program O, and fitting was improved by MAVE
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: DENSITY CORRELATION

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