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- PDB-6wcj: Asymmetric vertex of the clathrin minicoat cage -

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Basic information

Entry
Database: PDB / ID: 6wcj
TitleAsymmetric vertex of the clathrin minicoat cage
Components
  • Clathrin heavy chain 1
  • Clathrin light chain B
KeywordsENDOCYTOSIS / Clathrin coated vesicle / clathrin heavy chain / clathrin light chain / clathrin cage
Function / homology
Function and homology information


postsynaptic endocytic zone cytoplasmic component / Retrograde neurotrophin signalling / Recycling pathway of L1 / WNT5A-dependent internalization of FZD4 / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / LDL clearance / Gap junction degradation / Formation of annular gap junctions / Golgi Associated Vesicle Biogenesis / RHOU GTPase cycle ...postsynaptic endocytic zone cytoplasmic component / Retrograde neurotrophin signalling / Recycling pathway of L1 / WNT5A-dependent internalization of FZD4 / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / LDL clearance / Gap junction degradation / Formation of annular gap junctions / Golgi Associated Vesicle Biogenesis / RHOU GTPase cycle / RHOV GTPase cycle / clathrin coat of trans-Golgi network vesicle / clathrin vesicle coat / Lysosome Vesicle Biogenesis / negative regulation of hyaluronan biosynthetic process / clathrin light chain binding / clathrin complex / clathrin coat / MHC class II antigen presentation / VLDLR internalisation and degradation / clathrin heavy chain binding / clathrin coat of coated pit / Cargo recognition for clathrin-mediated endocytosis / clathrin coat assembly / clathrin coat disassembly / clathrin-coated endocytic vesicle / membrane coat / Clathrin-mediated endocytosis / clathrin-dependent endocytosis / arrestin family protein binding / ciliary membrane / receptor-mediated endocytosis / intracellular protein transport / trans-Golgi network / synaptic vesicle membrane / spindle / autophagy / disordered domain specific binding / melanosome / mitotic cell cycle / cell division / protein domain specific binding / structural molecule activity / mitochondrion / identical protein binding / plasma membrane / cytosol
Similarity search - Function
Clathrin light chain / Clathrin light chain signature 1. / Clathrin light chain signature 2. / Clathrin light chain / Clathrin-H-link / Clathrin, heavy chain, linker, core motif / Clathrin heavy chain, N-terminal / Clathrin, heavy chain / Clathrin, heavy chain, propeller repeat / Clathrin propeller repeat ...Clathrin light chain / Clathrin light chain signature 1. / Clathrin light chain signature 2. / Clathrin light chain / Clathrin-H-link / 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. / Tetratricopeptide-like helical domain superfamily / Armadillo-type fold
Similarity search - Domain/homology
Clathrin light chain B / Clathrin heavy chain 1
Similarity search - Component
Biological speciesBos taurus (cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.3 Å
AuthorsParaan, M. / Mendez, J. / Sharum, S. / Kurtin, D. / He, H. / Stagg, S.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM108753 United States
CitationJournal: Sci Adv / Year: 2020
Title: The structures of natively assembled clathrin-coated vesicles.
Authors: Mohammadreza Paraan / Joshua Mendez / Savanna Sharum / Danielle Kurtin / Huan He / Scott M Stagg /
Abstract: Clathrin-coated vesicles mediate trafficking of proteins and nutrients in the cell and between organelles. Proteins included in the clathrin-coated vesicles (CCVs) category include clathrin heavy ...Clathrin-coated vesicles mediate trafficking of proteins and nutrients in the cell and between organelles. Proteins included in the clathrin-coated vesicles (CCVs) category include clathrin heavy chain (CHC), clathrin light chain (CLC), and a variety of adaptor protein complexes. Much is known about the structures of the individual CCV components, but data are lacking about the structures of the fully assembled complexes together with membrane and in complex with cargo. Here, we determined the structures of natively assembled CCVs in a variety of geometries. We show that the adaptor β2 appendages crosslink adjacent CHC β-propellers and that the appendage densities are enriched in CCV hexagonal faces. We resolve how adaptor protein 2 and other associated factors in hexagonal faces form an assembly hub with an extensive web of interactions between neighboring β-propellers and propose a structural model that explains how adaptor binding can direct the formation of pentagonal and hexagonal faces.
History
DepositionMar 30, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 19, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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  • Simplified surface model + fitted atomic model
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Clathrin heavy chain 1
B: Clathrin light chain B
C: Clathrin heavy chain 1
E: Clathrin light chain B
D: Clathrin heavy chain 1
F: Clathrin light chain B
O: Clathrin light chain B
L: Clathrin heavy chain 1
M: Clathrin heavy chain 1
J: Clathrin light chain B
G: Clathrin heavy chain 1
H: Clathrin heavy chain 1
N: Clathrin light chain B
I: Clathrin heavy chain 1
K: Clathrin heavy chain 1


Theoretical massNumber of molelcules
Total (without water)1,876,85915
Polymers1,876,85915
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry, Using mass spectrometry, the presence of adaptors necessary for the assembly of clathrin coated vesicles in vivo was confirmed.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area50200 Å2
ΔGint-114 kcal/mol
Surface area264910 Å2

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Components

#1: Antibody
Clathrin heavy chain 1


Mass: 191800.312 Da / Num. of mol.: 9 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / References: UniProt: P49951
#2: Protein
Clathrin light chain B / Lcb


Mass: 25109.396 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / References: UniProt: P04975

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Natively assembled clathrin coated vesicles / Type: COMPLEX / Entity ID: all / Source: NATURAL
Source (natural)Organism: Bos taurus (cattle) / Organ: brain
Buffer solutionpH: 6.7
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMMES1
21 mMMagnesium ChlorideMgCl21
31 mMEGTA1
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: C-flat-2/2 4C
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 35000 X / Nominal defocus max: 5000 nm / Nominal defocus min: 3000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 800

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.17_3644refinement
PHENIX1.17_3644refinement
EM software
IDNameVersionCategoryFitting-ID
1RELION3particle selection
2Leginonimage acquisition
4cisTEM1CTF correction
5CTFFIND4CTF correction
6RELION3CTF correction
9Rosettamodel fitting1
11cryoSPARC2initial Euler assignment
12cisTEM1final Euler assignment
13RELION3final Euler assignment
15cisTEM13D reconstruction
35PHENIXmodel refinement1
63Rosettamodel fitting2
64PHENIXmodel refinement2
92Rosettamodel fitting3
93PHENIXmodel refinement3
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 360000
Details: These are subparticle images that were extracted from particle images. Each minicoat particle has 24 asymmetric vertices. From a total of 15000 minicoat particles, 360000 vertex subparticles ...Details: These are subparticle images that were extracted from particle images. Each minicoat particle has 24 asymmetric vertices. From a total of 15000 minicoat particles, 360000 vertex subparticles were extracted. The position of subparticles was calculated by the localized reconstruction script in scipion, and the subparticle images were extracted with RELION extraction tools.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 6.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 305413
Details: After the final refinement in cisTEM, half-maps were generated in cisTEM and the FSC was calculated with EMAN FSC tools.
Symmetry type: POINT
Atomic model building
IDProtocol
1AB INITIO MODEL
2AB INITIO MODEL
3FLEXIBLE FIT
Atomic model building

Accession code: 6SCT / Initial refinement model-ID: 1 / PDB-ID: 6SCT

6sct

/ Source name: PDB / Type: experimental model

IDPdb chain-ID 3D fitting-IDPdb chain residue range
1A11627-1641
2C11627-1641
3D11627-1641
4B2188-228
5E2188-228
6F2188-228
7A31248-1626
8C31248-1626
9D31248-1626
10B399-166
11E399-166
12F399-166
13O3
14L3
15M3
16J3
17G3
18H3
19N3
20I3
21K3
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 78.06 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.001241997
ELECTRON MICROSCOPYf_angle_d0.339356766
ELECTRON MICROSCOPYf_chiral_restr0.03286237
ELECTRON MICROSCOPYf_plane_restr0.00287359
ELECTRON MICROSCOPYf_dihedral_angle_d6.818915978

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