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- PDB-6yah: AP2 in clathrin coats assembled on a membrane containing dileucin... -

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Basic information

Entry
Database: PDB / ID: 6yah
TitleAP2 in clathrin coats assembled on a membrane containing dileucine- and tyrosine-based cargo peptides
Components
  • AP-2 complex subunit alpha-2
  • AP-2 complex subunit beta
  • AP-2 complex subunit mu
  • AP-2 complex subunit sigma
KeywordsENDOCYTOSIS / clathrin / clathrin adaptor / ap2 / clathrin assembly
Function / homology
Function and homology information


Gap junction degradation / Formation of annular gap junctions / LDL clearance / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / WNT5A-dependent internalization of FZD4 / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / LDL clearance / Retrograde neurotrophin signalling / Nef Mediated CD8 Down-regulation / Trafficking of GluR2-containing AMPA receptors ...Gap junction degradation / Formation of annular gap junctions / LDL clearance / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / WNT5A-dependent internalization of FZD4 / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / LDL clearance / Retrograde neurotrophin signalling / Nef Mediated CD8 Down-regulation / Trafficking of GluR2-containing AMPA receptors / VLDLR internalisation and degradation / Retrograde neurotrophin signalling / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / Trafficking of GluR2-containing AMPA receptors / WNT5A-dependent internalization of FZD4 / VLDLR internalisation and degradation / extrinsic component of presynaptic endocytic zone membrane / clathrin adaptor complex / WNT5A-dependent internalization of FZD4 / Recycling pathway of L1 / MHC class II antigen presentation / AP-2 adaptor complex / regulation of vesicle size / postsynaptic neurotransmitter receptor internalization / Cargo recognition for clathrin-mediated endocytosis / Recycling pathway of L1 / Retrograde neurotrophin signalling / clathrin-coated endocytic vesicle / Clathrin-mediated endocytosis / positive regulation of synaptic vesicle endocytosis / Cargo recognition for clathrin-mediated endocytosis / clathrin adaptor activity / Clathrin-mediated endocytosis / LDL clearance / membrane coat / vesicle budding from membrane / clathrin-dependent endocytosis / MHC class II antigen presentation / signal sequence binding / coronary vasculature development / Nef Mediated CD4 Down-regulation / endolysosome membrane / aorta development / ventricular septum development / Neutrophil degranulation / clathrin binding / low-density lipoprotein particle receptor binding / Trafficking of GluR2-containing AMPA receptors / Recycling pathway of L1 / positive regulation of receptor internalization / synaptic vesicle endocytosis / EPH-ephrin mediated repulsion of cells / negative regulation of protein localization to plasma membrane / vesicle-mediated transport / clathrin-coated pit / protein serine/threonine kinase binding / phosphatidylinositol binding / MHC class II antigen presentation / VLDLR internalisation and degradation / kidney development / intracellular protein transport / clathrin-coated endocytic vesicle membrane / terminal bouton / receptor internalization / cytoplasmic side of plasma membrane / kinase binding / disordered domain specific binding / endocytic vesicle membrane / synaptic vesicle / Cargo recognition for clathrin-mediated endocytosis / presynapse / Clathrin-mediated endocytosis / protein-containing complex assembly / cytoplasmic vesicle / Potential therapeutics for SARS / transmembrane transporter binding / postsynapse / protein domain specific binding / synapse / lipid binding / protein-containing complex binding / protein kinase binding / glutamatergic synapse / membrane / plasma membrane / cytosol
Similarity search - Function
AP-2 complex subunit sigma / Clathrin adaptor, alpha-adaptin, appendage, C-terminal subdomain / Adaptor protein complex AP-2, alpha subunit / Alpha adaptin AP2, C-terminal domain / Mu2, C-terminal domain / AP-2 complex subunit mu, N-terminal / Clathrin adaptor, beta-adaptin, appendage, Ig-like subdomain / Beta2-adaptin appendage, C-terminal sub-domain / AP-1/2/4 complex subunit beta / Adaptor protein complex, sigma subunit ...AP-2 complex subunit sigma / Clathrin adaptor, alpha-adaptin, appendage, C-terminal subdomain / Adaptor protein complex AP-2, alpha subunit / Alpha adaptin AP2, C-terminal domain / Mu2, C-terminal domain / AP-2 complex subunit mu, N-terminal / Clathrin adaptor, beta-adaptin, appendage, Ig-like subdomain / Beta2-adaptin appendage, C-terminal sub-domain / AP-1/2/4 complex subunit beta / Adaptor protein complex, sigma subunit / : / Beta-adaptin appendage, C-terminal subdomain / Beta2-adaptin appendage, C-terminal sub-domain / AP complex subunit beta / Clathrin adaptor complex, small chain / Clathrin adaptor complexes small chain signature. / Clathrin adaptor complexes medium chain signature 1. / Clathrin adaptor, mu subunit / Clathrin adaptor, mu subunit, conserved site / Clathrin adaptor complexes medium chain signature 2. / : / 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 / AP complex, mu/sigma subunit / Clathrin adaptor complex small chain / Adaptor complexes medium subunit family / AP-2 complex subunit mu, C-terminal superfamily / Clathrin/coatomer adaptor, adaptin-like, N-terminal / Adaptin N terminal region / Mu homology domain / Mu homology domain (MHD) profile. / Clathrin adaptor, appendage, Ig-like subdomain superfamily / Longin-like domain superfamily / TBP domain superfamily / Armadillo/beta-catenin-like repeats / Armadillo / Armadillo-like helical / Armadillo-type fold
Similarity search - Domain/homology
AP-2 complex subunit alpha-2 / AP-2 complex subunit sigma / AP-2 complex subunit beta / AP-2 complex subunit mu
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
Homo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 10.2 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
Revision 1.3May 22, 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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Assembly

Deposited unit
A: AP-2 complex subunit alpha-2
B: AP-2 complex subunit beta
M: AP-2 complex subunit mu
S: AP-2 complex subunit sigma


Theoretical massNumber of molelcules
Total (without water)244,0124
Polymers244,0124
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: equilibrium centrifugation, liposome pelleting assay
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein AP-2 complex subunit alpha-2 / 100 kDa coated vesicle protein C / Adaptor protein complex AP-2 subunit alpha-2 / Adaptor-related ...100 kDa coated vesicle protein C / Adaptor protein complex AP-2 subunit alpha-2 / Adaptor-related protein complex 2 subunit alpha-2 / Alpha-adaptin C / Alpha2-adaptin / Clathrin assembly protein complex 2 alpha-C large chain / Plasma membrane adaptor HA2/AP2 adaptin alpha C subunit


Mass: 70310.062 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Ap2a2, Adtab / Production host: Escherichia coli (E. coli) / References: UniProt: P18484
#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: 105619.344 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 (E. coli) / References: UniProt: P63010
#3: Protein AP-2 complex subunit mu / AP-2 mu chain / Adaptor protein complex AP-2 subunit mu / Adaptor-related protein complex 2 subunit ...AP-2 mu chain / Adaptor protein complex AP-2 subunit mu / Adaptor-related protein complex 2 subunit mu / Clathrin assembly protein complex 2 mu medium chain / Clathrin coat assembly protein AP50 / Clathrin coat-associated protein AP50 / Mu2-adaptin / Plasma membrane adaptor AP-2 50 kDa protein


Mass: 51044.113 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Ap2m1 / Production host: Escherichia coli (E. coli) / References: UniProt: P84092
#4: Protein AP-2 complex subunit sigma / Adaptor protein complex AP-2 subunit sigma / Adaptor-related protein complex 2 subunit sigma / ...Adaptor protein complex AP-2 subunit sigma / Adaptor-related protein complex 2 subunit sigma / Clathrin assembly protein 2 sigma small chain / Clathrin coat assembly protein AP17 / Clathrin coat-associated protein AP17 / Plasma membrane adaptor AP-2 17 kDa protein / Sigma-adaptin 3b / Sigma2-adaptin


Mass: 17038.688 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ap2s1, Ap17, Claps2 / Production host: Escherichia coli (E. coli) / References: UniProt: P62743

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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 AP2 membrane.COMPLEXClathrin/AP coat was formed on the membrane containing dileucine- and tyrosine-based cargo signal peptides. The AP2 adaptor lacked hinge and appendage regions in its alpha subunit.all0RECOMBINANT
2AP-2 complex in clathrin coat, subunits alpha-2 and muCOMPLEXAP2 in clathrin coat formed on membranes containing dileucine- and tyrosine-based cargo peptides#1, #31RECOMBINANT
3AP-2 complex in clathrin coat, subunit betaCOMPLEXAP2 in clathrin coat formed on membranes containing dileucine- and tyrosine-based cargo peptides#21RECOMBINANT
4AP-2 complex in clathrin coat, subunit sigmaCOMPLEXAP2 in clathrin coat formed on membranes containing dileucine- and tyrosine-based cargo peptides#41RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Rattus norvegicus (Norway rat)10116
23Homo sapiens (human)9606
34Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23Escherichia coli (E. coli)562
34Escherichia coli (E. coli)562
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 FIELD / 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
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: 10.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 27630 / Symmetry type: POINT
EM volume selectionMethod: geometrically defined initial positions on membrane strctures
Num. of tomograms: 29139 / Num. of volumes extracted: 292224 / Reference model: reference-free
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: correlation coefficient
Atomic model buildingPDB-ID: 1XA7

1xa7


Accession code: 1XA7 / Source name: PDB / Type: experimental model

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