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- EMDB-12984: Cryo-EM structure of the hub of the 28 triskelia mini clathrin co... -

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Basic information

Entry
Database: EMDB / ID: EMD-12984
TitleCryo-EM structure of the hub of the 28 triskelia mini clathrin coat complex, class 15
Map data
Sample
  • Complex: Beta2 appendage domain of AP2 bound to terminal domains beneath the hub of the 28 triskelia mini clathrin coat complex, class 15
    • Complex: Clathrin heavy chain
      • Protein or peptide: Clathrin heavy chain
    • Complex: AP-2 complex subunit beta
      • Protein or peptide: AP-2 complex subunit beta
Function / homology
Function and homology information


clathrin coat of trans-Golgi network vesicle / clathrin light chain binding / clathrin complex / Nef Mediated CD8 Down-regulation / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / clathrin adaptor complex / Trafficking of GluR2-containing AMPA receptors / WNT5A-dependent internalization of FZD4 / clathrin coat of coated pit / AP-2 adaptor complex ...clathrin coat of trans-Golgi network vesicle / clathrin light chain binding / clathrin complex / Nef Mediated CD8 Down-regulation / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / clathrin adaptor complex / Trafficking of GluR2-containing AMPA receptors / WNT5A-dependent internalization of FZD4 / clathrin coat of coated pit / AP-2 adaptor complex / postsynaptic neurotransmitter receptor internalization / Retrograde neurotrophin signalling / clathrin-coated endocytic vesicle / LDL clearance / clathrin-dependent endocytosis / coronary vasculature development / signal sequence binding / endolysosome membrane / Nef Mediated CD4 Down-regulation / aorta development / ventricular septum development / clathrin binding / Recycling pathway of L1 / synaptic vesicle endocytosis / EPH-ephrin mediated repulsion of cells / vesicle-mediated transport / MHC class II antigen presentation / VLDLR internalisation and degradation / kidney development / clathrin-coated endocytic vesicle membrane / intracellular protein transport / cytoplasmic side of plasma membrane / endocytic vesicle membrane / Cargo recognition for clathrin-mediated endocytosis / presynapse / Clathrin-mediated endocytosis / postsynapse / Potential therapeutics for SARS / glutamatergic synapse / structural molecule activity / membrane / plasma membrane / cytosol
Similarity search - Function
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 / Region in Clathrin and VPS / Clathrin heavy chain repeat homology / Clathrin, heavy chain/VPS, 7-fold repeat / Clathrin heavy-chain (CHCR) repeat profile. ...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 / Region in Clathrin and VPS / Clathrin heavy chain repeat homology / Clathrin, heavy chain/VPS, 7-fold repeat / Clathrin heavy-chain (CHCR) repeat profile. / Clathrin adaptor, beta-adaptin, appendage, Ig-like subdomain / Beta-adaptin appendage, C-terminal subdomain / AP-1/2/4 complex subunit beta / Beta2-adaptin appendage, C-terminal sub-domain / Beta2-adaptin appendage, C-terminal sub-domain / AP complex subunit beta / Coatomer/calthrin adaptor appendage, C-terminal subdomain / Clathrin adaptor, alpha/beta/gamma-adaptin, appendage, Ig-like subdomain / Adaptin C-terminal domain / Adaptin C-terminal domain / Clathrin/coatomer adaptor, adaptin-like, N-terminal / Adaptin N terminal region / Clathrin adaptor, appendage, Ig-like subdomain superfamily / TBP domain superfamily / Armadillo/beta-catenin-like repeats / Armadillo / Armadillo-like helical / Tetratricopeptide-like helical domain superfamily / Armadillo-type fold
Similarity search - Domain/homology
Clathrin heavy chain / AP-2 complex subunit beta
Similarity search - Component
Biological speciesSus scrofa (pig) / Homo sapiens (human) / pig (pig)
Methodsingle particle reconstruction / cryo EM / Resolution: 10.5 Å
AuthorsSmith SM / Smith CJ
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/N008391/1 United Kingdom
CitationJournal: EMBO J / Year: 2021
Title: Multi-modal adaptor-clathrin contacts drive coated vesicle assembly.
Authors: Sarah M Smith / Gabrielle Larocque / Katherine M Wood / Kyle L Morris / Alan M Roseman / Richard B Sessions / Stephen J Royle / Corinne J Smith /
Abstract: Clathrin-coated pits are formed by the recognition of membrane and cargo by the AP2 complex and the subsequent recruitment of clathrin triskelia. A role for AP2 in coated-pit assembly beyond initial ...Clathrin-coated pits are formed by the recognition of membrane and cargo by the AP2 complex and the subsequent recruitment of clathrin triskelia. A role for AP2 in coated-pit assembly beyond initial clathrin recruitment has not been explored. Clathrin binds the β2 subunit of AP2, and several binding sites have been identified, but our structural knowledge of these interactions is incomplete and their functional importance during endocytosis is unclear. Here, we analysed the cryo-EM structure of clathrin cages assembled in the presence of β2 hinge-appendage (β2HA). We find that the β2-appendage binds in at least two positions in the cage, demonstrating that multi-modal binding is a fundamental property of clathrin-AP2 interactions. In one position, β2-appendage cross-links two adjacent terminal domains from different triskelia. Functional analysis of β2HA-clathrin interactions reveals that endocytosis requires two clathrin interaction sites: a clathrin-box motif on the hinge and the "sandwich site" on the appendage. We propose that β2-appendage binding to more than one triskelion is a key feature of the system and likely explains why assembly is driven by AP2.
History
DepositionMay 21, 2021-
Header (metadata) releaseAug 11, 2021-
Map releaseAug 11, 2021-
UpdateJul 13, 2022-
Current statusJul 13, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0149
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0149
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7om8
  • Surface level: 0.016
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7om8
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12984.png

Downloads & links

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Map

FileDownload / File: emd_12984.map.gz / Format: CCP4 / Size: 163.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 2.78 Å
Density
Contour LevelBy AUTHOR: 0.0149 / Movie #1: 0.0149
Minimum - Maximum-0.053792544 - 0.12319828
Average (Standard dev.)0.00018319282 (±0.003739683)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions350350350
Spacing350350350
CellA=B=C: 973.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.782.782.78
M x/y/z350350350
origin x/y/z0.0000.0000.000
length x/y/z973.000973.000973.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS350350350
D min/max/mean-0.0540.1230.000

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Supplemental data

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Mask #1

Fileemd_12984_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_12984_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_12984_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Beta2 appendage domain of AP2 bound to terminal domains beneath t...

EntireName: Beta2 appendage domain of AP2 bound to terminal domains beneath the hub of the 28 triskelia mini clathrin coat complex, class 15
Components
  • Complex: Beta2 appendage domain of AP2 bound to terminal domains beneath the hub of the 28 triskelia mini clathrin coat complex, class 15
    • Complex: Clathrin heavy chain
      • Protein or peptide: Clathrin heavy chain
    • Complex: AP-2 complex subunit beta
      • Protein or peptide: AP-2 complex subunit beta

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Supramolecule #1: Beta2 appendage domain of AP2 bound to terminal domains beneath t...

SupramoleculeName: Beta2 appendage domain of AP2 bound to terminal domains beneath the hub of the 28 triskelia mini clathrin coat complex, class 15
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: Clathrin heavy chain

SupramoleculeName: Clathrin heavy chain / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Sus scrofa (pig)

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Supramolecule #3: AP-2 complex subunit beta

SupramoleculeName: AP-2 complex subunit beta / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Clathrin heavy chain

MacromoleculeName: Clathrin heavy chain / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: pig (pig)
Molecular weightTheoretical: 33.535574 KDa
SequenceString: MAQILPIRFQ EHLQLQNLGI NPANIGFSTL TMESDKFICI REKVGEQAQV VIIDMNDPSN PIRRPISADS AIMNPASKVI ALKAGKTLQ IFNIEMKSKM KAHTMTDDVT FWKWISLNTV ALVTDNAVYH WSMEGESQPV KMFDRHSSLA GCQIINYRTD A KQKWLLLT ...String:
MAQILPIRFQ EHLQLQNLGI NPANIGFSTL TMESDKFICI REKVGEQAQV VIIDMNDPSN PIRRPISADS AIMNPASKVI ALKAGKTLQ IFNIEMKSKM KAHTMTDDVT FWKWISLNTV ALVTDNAVYH WSMEGESQPV KMFDRHSSLA GCQIINYRTD A KQKWLLLT GISAQQNRVV GAMQLYSVDR KVSQPIEGHA ASFAQFKMEG NAEESTLFCF AVRGQAGGKL HIIEVGTPPT GN QPFPKKA VDVFFPPEAQ NDFPVAMQIS EKHDVVFLIT KYGYIHLYDL ETGTCIYMNR IS

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Macromolecule #2: AP-2 complex subunit beta

MacromoleculeName: AP-2 complex subunit beta / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 26.429457 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GGYVAPKAVW LPAVKAKGLE ISGTFTHRQG HIYMEMNFTN KALQHMTDFA IQFNKNSFGV IPSTPLAIHT PLMPNQSIDV SLPLNTLGP VMKMEPLNNL QVAVKNNIDV FYFSCLIPLN VLFVEDGKME RQVFLATWKD IPNENELQFQ IKECHLNADT V SSKLQNNN ...String:
GGYVAPKAVW LPAVKAKGLE ISGTFTHRQG HIYMEMNFTN KALQHMTDFA IQFNKNSFGV IPSTPLAIHT PLMPNQSIDV SLPLNTLGP VMKMEPLNNL QVAVKNNIDV FYFSCLIPLN VLFVEDGKME RQVFLATWKD IPNENELQFQ IKECHLNADT V SSKLQNNN VYTIAKRNVE GQDMLYQSLK LTNGIWILAE LRIQPGNPNY TLSLKCRAPE VSQYIYQVYD SILKN

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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: 6.4
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 90 % / Chamber temperature: 277.15 K / Instrument: LEICA EM GP
Details: 3 uL of sample applied to a grid and blotted for 4.5 s before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.1 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Average electron dose: 64.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: RELION (ver. 3.0.5)
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 10.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 11676
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsThis structural model has been updated to include only clathrin terminal domain beta propeller residues that are close to the interface with the beta2 appendage domain.
Output model

PDB-7om8:
Beta2 appendage domain of AP2 bound to terminal domains beneath the hub of the 28 triskelia mini clathrin coat complex, class 15

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