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- PDB-6yai: Clathrin with bound beta2 appendage of AP2 -

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Basic information

Entry
Database: PDB / ID: 6yai
TitleClathrin with bound beta2 appendage of AP2
Components
  • (Clathrin heavy chain) x 2
  • AP-2 complex subunit beta
  • Clathrin light chain
KeywordsENDOCYTOSIS / clathrin / clathrin adaptor / ap2 / clathrin assembly
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 / clathrin-coated vesicle / 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 / disordered domain specific binding / Cargo recognition for clathrin-mediated endocytosis / presynapse / Clathrin-mediated endocytosis / postsynapse / Potential therapeutics for SARS / glutamatergic synapse / structural molecule activity / nucleolus / nucleoplasm / membrane / 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. / Ribosome biogenesis protein Nop16 / Ribosome biogenesis protein Nop16 / 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 light chain / Clathrin heavy chain / Nucleolar protein 16 / Clathrin heavy chain / AP-2 complex subunit beta
Similarity search - Component
Biological speciesHomo sapiens (human)
Sus scrofa (pig)
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 9.2 Å
AuthorsKovtun, O. / Kane Dickson, V. / Kelly, B.T. / Owen, D. / Briggs, J.A.G.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Wellcome Trust207455/Z/17/Z United Kingdom
Medical Research Council (MRC, United Kingdom)MC_UP_1201/16 United Kingdom
CitationJournal: Sci Adv / Year: 2020
Title: Architecture of the AP2/clathrin coat on the membranes of clathrin-coated vesicles.
Authors: Oleksiy Kovtun / Veronica Kane Dickson / Bernard T Kelly / David J Owen / John A G Briggs /
Abstract: Clathrin-mediated endocytosis (CME) is crucial for modulating the protein composition of a cell's plasma membrane. Clathrin forms a cage-like, polyhedral outer scaffold around a vesicle, to which ...Clathrin-mediated endocytosis (CME) is crucial for modulating the protein composition of a cell's plasma membrane. Clathrin forms a cage-like, polyhedral outer scaffold around a vesicle, to which cargo-selecting clathrin adaptors are attached. Adaptor protein complex (AP2) is the key adaptor in CME. Crystallography has shown AP2 to adopt a range of conformations. Here, we used cryo-electron microscopy, tomography, and subtomogram averaging to determine structures, interactions, and arrangements of clathrin and AP2 at the key steps of coat assembly, from AP2 in solution to membrane-assembled clathrin-coated vesicles (CCVs). AP2 binds cargo and PtdIns(4,5) (phosphatidylinositol 4,5-bisphosphate)-containing membranes via multiple interfaces, undergoing conformational rearrangement from its cytosolic state. The binding mode of AP2 β2 appendage into the clathrin lattice in CCVs and buds implies how the adaptor structurally modulates coat curvature and coat disassembly.
History
DepositionMar 12, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 29, 2020Provider: repository / Type: Initial release
Revision 1.1Aug 12, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Aug 19, 2020Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
E: Clathrin heavy chain
F: AP-2 complex subunit beta
C: Clathrin heavy chain
M: Clathrin heavy chain
A: Clathrin heavy chain
B: Clathrin heavy chain
D: Clathrin light chain
O: Clathrin light chain
J: Clathrin heavy chain
K: Clathrin heavy chain
L: Clathrin heavy chain
H: Clathrin heavy chain
I: Clathrin light chain
N: Clathrin light chain


Theoretical massNumber of molelcules
Total (without water)1,811,58214
Polymers1,811,58214
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: equilibrium centrifugation, liposome pull-down assay was used to confirm the assembly.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area20400 Å2
ΔGint-89 kcal/mol
Surface area228130 Å2
MethodPISA

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Components

#1: Antibody
Clathrin heavy chain /


Mass: 187145.125 Da / Num. of mol.: 8 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / References: UniProt: C0MHR2, UniProt: I3LGD4*PLUS
#2: Protein AP-2 complex subunit beta / AP105B / Adaptor protein complex AP-2 subunit beta / Adaptor-related protein complex 2 subunit beta ...AP105B / Adaptor protein complex AP-2 subunit beta / Adaptor-related protein complex 2 subunit beta / Beta-2-adaptin / Beta-adaptin / Clathrin assembly protein complex 2 beta large chain / Plasma membrane adaptor HA2/AP2 adaptin beta subunit


Mass: 26429.457 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: AP2B1, ADTB2, CLAPB1
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P63010
#3: Antibody Clathrin heavy chain /


Mass: 187117.094 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / References: UniProt: C0MHR2, UniProt: I3LGD4*PLUS
#4: Protein
Clathrin light chain /


Mass: 25218.500 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / References: UniProt: F1S398, UniProt: A0A480JRB0*PLUS

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: subtomogram averaging

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Clathrin/AP2 coat assembled on membrane.COMPLEXClathrin/AP coat was formed on the membrane containing cargo signal peptides. The AP2 adaptor lacked hinge and appendage regions in its alpha subunit.all0MULTIPLE SOURCES
2ClathrinCOMPLEXClathrin in the clathrin/AP2 coat formed on the membrane#1, #3-#41NATURAL
3beta2 appendage of AP2 adaptorCOMPLEXbeta2 appendage of the AP2 bound to clathrin to clathrin/AP2 coat formed on the membrane#21RECOMBINANT
Molecular weight
IDEntity assembly-IDUnitsExperimental value
11MEGADALTONSNO
21MEGADALTONSNO
31MEGADALTONSNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDOrgan
12Sus scrofa (pig)9823brain
23Homo sapiens (human)9606
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 7.2
Buffer component
IDConc.NameBuffer-ID
110 mMHepes KOH1
2120 mMPotassium Acetate1
32 mMMagnesium chloride1
45 mM2-Mercaptoethanol1
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The sample (in vitro budding reaction) contained AP2, clathrin and 400 nm extruded liposomes
Specimen supportGrid type: C-flat-2/2
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 98 % / Chamber temperature: 291 K
Details: The sample was supplemented with 10 nm nanogold fiducials, and 3 ul of the mixture was backside blotted for 3 seconds.

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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: 81000 X / Calibrated magnification: 81000 X / Nominal defocus max: 6500 nm / Nominal defocus min: 1500 nm / Calibrated defocus min: 1500 nm / Calibrated defocus max: 6500 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 0.2 sec. / Electron dose: 3.2 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 2
Details: The images were collected in movie mode at 10 frames per second
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 10 / Used frames/image: 1-10

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Processing

EM software
IDNameVersionCategory
1TOMvolume selection
2SerialEM3.6image acquisition
4NOVACTF1CTF correction
7MDFF1.7model fitting
10TOMfinal Euler assignment
11AV3final Euler assignment
12PEETclassification
13Dynamoclassification
14TOM3D reconstruction
15AV33D reconstruction
16PHENIXmodel refinement
Image processingDetails: The images were low pass filtered according to the cumulative radiation dose.
CTF correctionDetails: CTF correction in novaCTF with by multiplication / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 9.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 12076 / Symmetry type: POINT
EM volume selectionMethod: geometrically defined initial positions
Details: To define initial positions of subtomograms, centres and radii of coated vesicles, buds were manually marked in bin4 tomograms. These measurements were used to define spheres. Subtomogram ...Details: To define initial positions of subtomograms, centres and radii of coated vesicles, buds were manually marked in bin4 tomograms. These measurements were used to define spheres. Subtomogram positions were then defined on the surface of these spheres with uniform sampling. The orientations were calculated to be normal to the surfaces of the spheres with random in-plane rotation.
Num. of tomograms: 58121 / Num. of volumes extracted: 198871 / Reference model: reference-free
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: correlation coefficient
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
11XI4

1xi4

1
26SCT

6sct

1
31.0E+42A1

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