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- EMDB-24710: AP2 bound to heparin in the closed conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-24710
TitleAP2 bound to heparin in the closed conformation
Map dataunsharpened, full map
Sample
  • Complex: AP2 complex bound to heparin
    • Protein or peptide: AP-2 complex subunit alpha-2
    • Protein or peptide: AP-2 complex subunit beta
    • Protein or peptide: AP-2 complex subunit mu
    • Protein or peptide: AP-2 complex subunit sigma
Function / homology
Function and homology information


Formation of annular gap junctions / Gap junction degradation / LDL clearance / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / WNT5A-dependent internalization of FZD4 / Trafficking of GluR2-containing AMPA receptors / Retrograde neurotrophin signalling / clathrin coat / clathrin adaptor complex / extrinsic component of presynaptic endocytic zone membrane ...Formation of annular gap junctions / Gap junction degradation / LDL clearance / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / WNT5A-dependent internalization of FZD4 / Trafficking of GluR2-containing AMPA receptors / Retrograde neurotrophin signalling / clathrin coat / clathrin adaptor complex / extrinsic component of presynaptic endocytic zone membrane / VLDLR internalisation and degradation / cardiac septum development / Recycling pathway of L1 / AP-2 adaptor complex / regulation of vesicle size / postsynaptic neurotransmitter receptor internalization / clathrin coat assembly / Cargo recognition for clathrin-mediated endocytosis / positive regulation of synaptic vesicle endocytosis / clathrin adaptor activity / Clathrin-mediated endocytosis / vesicle budding from membrane / membrane coat / clathrin-dependent endocytosis / MHC class II antigen presentation / protein serine/threonine kinase binding / neurotransmitter receptor internalization / positive regulation of protein localization to membrane / coronary vasculature development / signal sequence binding / negative regulation of protein localization to plasma membrane / aorta development / regulation of hematopoietic stem cell differentiation / ventricular septum development / low-density lipoprotein particle receptor binding / clathrin binding / positive regulation of endocytosis / positive regulation of receptor internalization / synaptic vesicle endocytosis / clathrin-coated pit / vesicle-mediated transport / Neutrophil degranulation / phosphatidylinositol binding / secretory granule / kidney development / intracellular protein transport / cytoplasmic side of plasma membrane / receptor internalization / kinase binding / disordered domain specific binding / synaptic vesicle / heart development / cytoplasmic vesicle / postsynapse / protein-containing complex assembly / transmembrane transporter binding / protein domain specific binding / intracellular membrane-bounded organelle / synapse / glutamatergic synapse / lipid binding / protein-containing complex binding / protein kinase binding / mitochondrion / plasma membrane
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 / Adaptor protein complex, sigma subunit / Clathrin adaptor, beta-adaptin, appendage, Ig-like subdomain / Beta-adaptin appendage, C-terminal subdomain / AP-1/2/4 complex subunit beta ...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 / Adaptor protein complex, sigma subunit / 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 / 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 / Adaptor complexes medium subunit family / AP complex, mu/sigma subunit / Clathrin adaptor complex small chain / AP-2 complex subunit mu, C-terminal superfamily / Mu homology domain / Mu homology domain (MHD) profile. / 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 / 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 mu / AP-2 complex subunit beta
Similarity search - Component
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsBaker RW / Hollopeter G / Partlow EA
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM127548-01A1 United States
CitationJournal: Nat Struct Mol Biol / Year: 2022
Title: Structural basis of an endocytic checkpoint that primes the AP2 clathrin adaptor for cargo internalization.
Authors: Edward A Partlow / Kevin S Cannon / Gunther Hollopeter / Richard W Baker /
Abstract: Clathrin-mediated endocytosis (CME) is the main route of internalization from the plasma membrane. It is known that the heterotetrameric AP2 clathrin adaptor must open to simultaneously engage ...Clathrin-mediated endocytosis (CME) is the main route of internalization from the plasma membrane. It is known that the heterotetrameric AP2 clathrin adaptor must open to simultaneously engage membrane and endocytic cargo, yet it is unclear how transmembrane cargos are captured to catalyze CME. Using cryogenic-electron microscopy, we discover a new way in which mouse AP2 can reorganize to expose membrane- and cargo-binding pockets, which is not observed in clathrin-coated structures. Instead, it is stimulated by endocytic pioneer proteins called muniscins, which do not enter vesicles. Muniscin-engaged AP2 is primed to rearrange into the vesicle-competent conformation on binding the tyrosine cargo internalization motif (YxxΦ). We propose adaptor priming as a checkpoint to ensure cargo internalization.
History
DepositionAug 19, 2021-
Header (metadata) releaseMar 30, 2022-
Map releaseMar 30, 2022-
UpdateApr 27, 2022-
Current statusApr 27, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_24710.png

Downloads & links

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Map

FileDownload / File: emd_24710.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationunsharpened, full map
Voxel sizeX=Y=Z: 0.89 Å
Density
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-0.4452418 - 1.0589159
Average (Standard dev.)0.00011173194 (±0.02781827)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 320.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_24710_msk_1.map
Projections & Slices
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Additional map: map filtered by local resolution

Fileemd_24710_additional_1.map
Annotationmap filtered by local resolution
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Additional map: local resolution file

Fileemd_24710_additional_2.map
Annotationlocal resolution file
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Additional map: sharpened map

Fileemd_24710_additional_3.map
Annotationsharpened map
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Half map: half map B

Fileemd_24710_half_map_1.map
Annotationhalf map B
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Half map: half map A

Fileemd_24710_half_map_2.map
Annotationhalf map A
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Sample components

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Entire : AP2 complex bound to heparin

EntireName: AP2 complex bound to heparin
Components
  • Complex: AP2 complex bound to heparin
    • Protein or peptide: AP-2 complex subunit alpha-2
    • Protein or peptide: AP-2 complex subunit beta
    • Protein or peptide: AP-2 complex subunit mu
    • Protein or peptide: AP-2 complex subunit sigma

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Supramolecule #1: AP2 complex bound to heparin

SupramoleculeName: AP2 complex bound to heparin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pACYC Duet; pET Duet

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Macromolecule #1: AP-2 complex subunit alpha-2

MacromoleculeName: AP-2 complex subunit alpha-2 / type: protein_or_peptide / ID: 1 / Details: AP2 alpha 1-621 purified with a C-terminal GST tag / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 70.57332 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MPAVSKGDGM RGLAVFISDI RNCKSKEAEI KRINKELANI RSKFKGDKAL DGYSKKKYVC KLLFIFLLGH DIDFGHMEAV NLLSSNRYT EKQIGYLFIS VLVNSNSELI RLINNAIKND LASRNPTFMG LALHCIANVG SREMAEAFAG EIPKILVAGD T MDSVKQSA ...String:
MPAVSKGDGM RGLAVFISDI RNCKSKEAEI KRINKELANI RSKFKGDKAL DGYSKKKYVC KLLFIFLLGH DIDFGHMEAV NLLSSNRYT EKQIGYLFIS VLVNSNSELI RLINNAIKND LASRNPTFMG LALHCIANVG SREMAEAFAG EIPKILVAGD T MDSVKQSA ALCLLRLYRT SPDLVPMGDW TSRVVHLLND QHLGVVTAAT SLITTLAQKN PEEFKTSVSL AVSRLSRIVT SA STDLQDY TYYFVPAPWL SVKLLRLLQC YPPPEDPAVR GRLTECLETI LNKAQEPPKS KKVQHSNAKN AVLFEAISLI IHH DSEPNL LVRACNQLGQ FLQHRETNLR YLALESMCTL ASSEFSHEAV KTHIETVINA LKTERDVSVR QRAVDLLYAM CDRS NAQQI VAEMLSYLET ADYSIREEIV LKVAILAEKY AVDYTWYVDT ILNLIRIAGD YVSEEVWYRV IQIVINRDDV QGYAA KTVF EALQAPACHE NLVKVGGYIL GEFGNLIAGD PRSSPLIQFN LLHSKFHLCS VPTRALLLST YIKFVNLFPE VKATIQ DVL RSDSQLKNAD VELQQRAVEY LRLSTVASTD ILATVLEEMP PFPERESSIL AKLKKKKGGS GLEVLFQ

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Macromolecule #2: AP-2 complex subunit beta

MacromoleculeName: AP-2 complex subunit beta / type: protein_or_peptide / ID: 2 / Details: AP2 beta 1-591 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 66.953195 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString: MTDSKYFTTN KKGEIFELKA ELNNEKKEKR KEAVKKVIAA MTVGKDVSSL FPDVVNCMQT DNLELKKLVY LYLMNYAKSQ PDMAIMAVN SFVKDCEDPN PLIRALAVRT MGCIRVDKIT EYLCEPLRKC LKDEDPYVRK TAAVCVAKLH DINAQMVEDQ G FLDSLRDL ...String:
MTDSKYFTTN KKGEIFELKA ELNNEKKEKR KEAVKKVIAA MTVGKDVSSL FPDVVNCMQT DNLELKKLVY LYLMNYAKSQ PDMAIMAVN SFVKDCEDPN PLIRALAVRT MGCIRVDKIT EYLCEPLRKC LKDEDPYVRK TAAVCVAKLH DINAQMVEDQ G FLDSLRDL IADSNPMVVA NAVAALSEIS ESHPNSNLLD LNPQNINKLL TALNECTEWG QIFILDCLSN YNPKDDREAQ SI CERVTPR LSHANSAVVL SAVKVLMKFL ELLPKDSDYY NMLLKKLAPP LVTLLSGEPE VQYVALRNIN LIVQKRPEIL KQE IKVFFV KYNDPIYVKL EKLDIMIRLA SQANIAQVLA ELKEYATEVD VDFVRKAVRA IGRCAIKVEQ SAERCVSTLL DLIQ TKVNY VVQEAIVVIR DIFRKYPNKY ESIIATLCEN LDSLDEPDAR AAMIWIVGEY AERIDNADEL LESFLEGFHD ESTQV QLTL LTAIVKLFLK KPSETQELVQ QVLSLATQDS DNPDLRDRGY IYWRLLSTDP VTAKEVVLSE KPLISEETDL IEPTLL DEL ICHIGSLASV YHKPPNAFVE GSHGIHRK

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Macromolecule #3: AP-2 complex subunit mu

MacromoleculeName: AP-2 complex subunit mu / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 49.726641 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MIGGLFIYNH KGEVLISRVY RDDIGRNAVD AFRVNVIHAR QQVRSPVTNI ARTSFFHVKR SNIWLAAVTK QNVNAAMVFE FLYKMCDVM AAYFGKISEE NIKNNFVLIY ELLDEILDFG YPQNSETGAL KTFITQQGIK SQHQTKEEQS QITSQVTGQI G WRREGIKY ...String:
MIGGLFIYNH KGEVLISRVY RDDIGRNAVD AFRVNVIHAR QQVRSPVTNI ARTSFFHVKR SNIWLAAVTK QNVNAAMVFE FLYKMCDVM AAYFGKISEE NIKNNFVLIY ELLDEILDFG YPQNSETGAL KTFITQQGIK SQHQTKEEQS QITSQVTGQI G WRREGIKY RRNELFLDVL ESVNLLMSPQ GQVLSAHVSG RVVMKSYLSG MPECKFGMND KIVIEKQGKG TADETSKSGK QS IAIDDCT FHQCVRLSKF DSERSISFIP PDGEFELMRY RTTKDIILPF RVIPLVREVG RTKLEVKVVI KSNFKPSLLA QKI EVRIPT PLNTSGVQVI CMKGKAKYKA SENAIVWKIK RMAGMKESQI SAEIELLPTN DKKKWARPPI SMNFEVPFAP SGLK VRYLK VFEPKLNYSD HDVIKWVRYI GRSGIYETRC

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Macromolecule #4: AP-2 complex subunit sigma

MacromoleculeName: AP-2 complex subunit sigma / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 17.038688 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MIRFILIQNR AGKTRLAKWY MQFDDDEKQK LIEEVHAVVT VRDAKHTNFV EFRNFKIIYR RYAGLYFCIC VDVNDNNLAY LEAIHNFVE VLNEYFHNVC ELDLVFNFYK VYTVVDEMFL AGEIRETSQT KVLKQLLMLQ SLE

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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

Concentration2 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chlorideSodium Chloride
0.05 %C14H28O6n-octyl-beta-d-glucoside
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Blot force -10, 4 second blot, 4 uL sample.
DetailsHeparin added at a 2-fold excess 20 mM HEPES pH 7.4, 150 mM NaCl, 1 mM DTT, 0.05% beta-OG

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 45000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 55.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 805304
CTF correctionSoftware - Name: cryoSPARC (ver. 3.2.0)
Startup modelType of model: OTHER
Details: ab initio model generation performed in cryoSPARC v3.2.0
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE / Software - Name: cryoSPARC (ver. 3.2.0)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.0) / Details: Non-Uniform refinement in cryoSPARC v3.2.0 / Number images used: 157670
DetailsCounting Mode
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

2vgl

Detailsphenix.real_space_refine
RefinementProtocol: RIGID BODY FIT
Output model

PDB-7rw8:
AP2 bound to heparin in the closed conformation

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