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- EMDB-24711: AP2 bound to heparin in the bowl conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-24711
TitleAP2 bound to heparin in the bowl conformation
Map dataunsharpened map
Sample
  • Complex: AP2 complex bound to heparin in the "bowl" conformation
    • 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
KeywordsAP2 / clathrin vesicle / endocytosis / lipid-binding / adaptor / membrane / transport
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 adaptor complex / clathrin coat / 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 adaptor complex / clathrin coat / 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 / Cargo recognition for clathrin-mediated endocytosis / clathrin coat assembly / Clathrin-mediated endocytosis / positive regulation of synaptic vesicle endocytosis / clathrin adaptor activity / membrane coat / vesicle budding from membrane / clathrin-dependent endocytosis / coronary vasculature development / MHC class II antigen presentation / positive regulation of protein localization to membrane / neurotransmitter receptor internalization / signal sequence binding / 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 / negative regulation of protein localization to plasma membrane / protein serine/threonine kinase binding / vesicle-mediated transport / Neutrophil degranulation / phosphatidylinositol binding / secretory granule / kidney development / intracellular protein transport / kinase binding / cytoplasmic side of plasma membrane / disordered domain specific binding / synaptic vesicle / heart development / protein-containing complex assembly / cytoplasmic vesicle / transmembrane transporter binding / postsynapse / protein domain specific binding / intracellular membrane-bounded organelle / lipid binding / glutamatergic synapse / synapse / protein-containing complex binding / protein kinase binding / mitochondrion
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 / Beta2-adaptin appendage, C-terminal sub-domain ...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 / Beta2-adaptin appendage, C-terminal sub-domain / Beta2-adaptin appendage, C-terminal sub-domain / AP-1/2/4 complex subunit beta / : / 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 / Clathrin adaptor, alpha/beta/gamma-adaptin, appendage, Ig-like subdomain / Mu homology domain / Adaptin C-terminal domain / Mu homology domain (MHD) profile. / 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: 3.9 Å
AuthorsBaker RW / Hollopeter G
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-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_24711.png

Downloads & links

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Map

FileDownload / File: emd_24711.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationunsharpened map
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.89 Å/pix.
x 360 pix.
= 320.4 Å		0.89 Å/pix.
x 360 pix.
= 320.4 Å		0.89 Å/pix.
x 360 pix.
= 320.4 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.89 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.17127171 - 0.43660972
Average (Standard dev.)-0.000057003177 (±0.010168169)
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_24711_msk_1.map
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Additional map: sharpened map

Fileemd_24711_additional_1.map
Annotationsharpened map
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Additional map: local resolution file

Fileemd_24711_additional_2.map
Annotationlocal resolution file
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Additional map: map filtered by local resolution

Fileemd_24711_additional_3.map
Annotationmap filtered by local resolution
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Half map: half map B

Fileemd_24711_half_map_1.map
Annotationhalf map B
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Half map: half map A

Fileemd_24711_half_map_2.map
Annotationhalf map A
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Sample components

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Entire : AP2 complex bound to heparin in the "bowl" conformation

EntireName: AP2 complex bound to heparin in the "bowl" conformation
Components
  • Complex: AP2 complex bound to heparin in the "bowl" conformation
    • 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 in the "bowl" conformation

SupramoleculeName: AP2 complex bound to heparin in the "bowl" conformation
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mus musculus (house mouse)

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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: Expressed as a C-terminal GST fusion and the tag was removed with Prescission protease
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

UniProtKB: AP-2 complex subunit alpha-2

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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: Mus musculus (house mouse)
Molecular weightTheoretical: 66.953195 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
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

UniProtKB: AP-2 complex subunit beta

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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

UniProtKB: AP-2 complex subunit mu

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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

UniProtKB: AP-2 complex subunit sigma

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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 Chloride
0.05 %C14H28O6n-octyl-beta-d-glucoside
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 60 sec. / 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
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 55.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 45000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

DetailsCounting Mode
Particle selectionNumber selected: 805304
Startup modelType of model: OTHER
Details: ab initio model generation performed in cryoSPARC v3.2.0
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.0) / Number images used: 151164
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE / Software - Name: cryoSPARC (ver. 3.2.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

2vgl


Chain - Source name: PDB / Chain - Initial model type: experimental model
Detailsphenix.real_space_refine
RefinementProtocol: RIGID BODY FIT
Output model

PDB-7rw9:
AP2 bound to heparin in the bowl conformation

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