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- EMDB-20215: CRYO-EM STRUCTURE OF PHOSPHORYLATED AP-2 CORE BOUND TO NECAP -

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Basic information

Entry
Database: EMDB / ID: EMD-20215
TitleCRYO-EM STRUCTURE OF PHOSPHORYLATED AP-2 CORE BOUND TO NECAP
Map datacryo-EM structure of phosphorylated AP2 bound to NECAP; sharpened map
Sample
  • Complex: PHOSPHORYLATED AP2-NECAP CO-COMPLEX
    • 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
    • Protein or peptide: Adaptin ear-binding coat-associated protein 2
KeywordsAP2 / NECAP2 PROTEIN / CLATHRIN VESICLE / ENDOCYTOSIS / LIPID-BINDING / ADAPTOR / MEMBRANE / TRANSPORT / PHOSPHORYLATION
Function / homology
Function and homology information


Formation of annular gap junctions / Gap junction degradation / clathrin vesicle coat / 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 / Trafficking of GluR2-containing AMPA receptors ...Formation of annular gap junctions / Gap junction degradation / clathrin vesicle coat / 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 / Trafficking of GluR2-containing AMPA receptors / VLDLR internalisation and degradation / Retrograde neurotrophin signalling / WNT5A-dependent internalization of FZD4 / VLDLR internalisation and degradation / clathrin adaptor complex / cardiac septum development / Recycling pathway of L1 / extrinsic component of presynaptic endocytic zone membrane / MHC class II antigen presentation / regulation of vesicle size / AP-2 adaptor complex / postsynaptic endocytic zone / postsynaptic neurotransmitter receptor internalization / Cargo recognition for clathrin-mediated endocytosis / Recycling pathway of L1 / membrane coat / Clathrin-mediated endocytosis / positive regulation of synaptic vesicle endocytosis / Cargo recognition for clathrin-mediated endocytosis / clathrin coat assembly / clathrin adaptor activity / Clathrin-mediated endocytosis / vesicle budding from membrane / clathrin-dependent endocytosis / MHC class II antigen presentation / signal sequence binding / coronary vasculature development / positive regulation of protein localization to membrane / neurotransmitter secretion / regulation of hematopoietic stem cell differentiation / aorta development / ventricular septum development / low-density lipoprotein particle receptor binding / clathrin binding / Trafficking of GluR2-containing AMPA receptors / positive regulation of endocytosis / positive regulation of receptor internalization / synaptic vesicle endocytosis / negative regulation of protein localization to plasma membrane / vesicle-mediated transport / Neutrophil degranulation / secretory granule / intracellular protein transport / kidney development / kinase binding / cytoplasmic side of plasma membrane / endocytosis / disordered domain specific binding / synaptic vesicle / protein transport / presynapse / heart development / protein-containing complex assembly / cytoplasmic vesicle / transmembrane transporter binding / postsynapse / protein domain specific binding / intracellular membrane-bounded organelle / lipid binding / synapse / protein kinase binding / protein-containing complex binding / glutamatergic synapse / mitochondrion / plasma membrane
Similarity search - Function
NECAP, PHear domain / Protein of unknown function (DUF1681) / 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 ...NECAP, PHear domain / Protein of unknown function (DUF1681) / 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 / Adaptor complexes medium subunit family / Clathrin adaptor complex small chain / 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 / PH-like domain superfamily / Armadillo-type fold
Similarity search - Domain/homology
AP-2 complex subunit alpha-2 / AP-2 complex subunit sigma / AP-2 complex subunit mu / Adaptin ear-binding coat-associated protein 2 / AP-2 complex subunit beta
Similarity search - Component
Biological speciesMus musculus (house mouse) / Rattus norvegicus (Norway rat)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsPartlow EA / Baker RW
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: Elife / Year: 2019
Title: A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex.
Authors: Partlow EA / Baker RW / Beacham GM / Chappie JS / Leschziner AE / Hollopeter G
History
DepositionMay 10, 2019-
Header (metadata) releaseAug 28, 2019-
Map releaseSep 11, 2019-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.03
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  • Surface view with fitted model
  • Atomic models: PDB-6owo
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol

Downloads & links

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Map

FileDownload / File: emd_20215.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcryo-EM structure of phosphorylated AP2 bound to NECAP; sharpened map
Voxel sizeX=Y=Z: 1.16 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.09064991 - 0.174634
Average (Standard dev.)0.000040236286 (±0.004779916)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 348.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.161.161.16
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z348.000348.000348.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0910.1750.000

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Supplemental data

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Sample components

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Entire : PHOSPHORYLATED AP2-NECAP CO-COMPLEX

EntireName: PHOSPHORYLATED AP2-NECAP CO-COMPLEX
Components
  • Complex: PHOSPHORYLATED AP2-NECAP CO-COMPLEX
    • 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
    • Protein or peptide: Adaptin ear-binding coat-associated protein 2

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Supramolecule #1: PHOSPHORYLATED AP2-NECAP CO-COMPLEX

SupramoleculeName: PHOSPHORYLATED AP2-NECAP CO-COMPLEX / 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 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 69.656297 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MPAVSKGEGM RGLAVFISDI RNCKSKEAEI KRINKELANI RSKFKGDKAL DGYSKKKYVC KLLFIFLLGH DIDFGHMEAV NLLSSNRYT EKQIGYLFIS VLVNSNSELI RLINNAIKND LASRNPTFMG LALHCIANVG SREMAEAFAG EIPKILVAGD T MDSVKQSA ...String:
MPAVSKGEGM 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 AKLKKKKG

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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: 49.806621 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MIGGLFIYNH KGEVLISRVY RDDIGRNAVD AFRVNVIHAR QQVRSPVTNI ARTSFFHVKR SNIWLAAVTK QNVNAAMVFE FLYKMCDVM AAYFGKISEE NIKNNFVLIY ELLDEILDFG YPQNSETGAL KTFITQQGIK SQHQTKEEQS QITSQV(TPO) GQ IGWRREGIKY ...String:
MIGGLFIYNH KGEVLISRVY RDDIGRNAVD AFRVNVIHAR QQVRSPVTNI ARTSFFHVKR SNIWLAAVTK QNVNAAMVFE FLYKMCDVM AAYFGKISEE NIKNNFVLIY ELLDEILDFG YPQNSETGAL KTFITQQGIK SQHQTKEEQS QITSQV(TPO) GQ IGWRREGIKY RRNELFLDVL ESVNLLMSPQ GQVLSAHVSG RVVMKSYLSG MPECKFGMND KIVIEKQGKG TADETSKS G KQSIAIDDCT FHQCVRLSKF DSERSISFIP PDGEFELMRY RTTKDIILPF RVIPLVREVG RTKLEVKVVI KSNFKPSLL AQKIEVRIPT PLNTSGVQVI CMKGKAKYKA SENAIVWKIK RMAGMKESQI SAEIELLPTN DKKKWARPPI SMNFEVPFAP SGLKVRYLK 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: Rattus norvegicus (Norway rat)
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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Macromolecule #5: Adaptin ear-binding coat-associated protein 2

MacromoleculeName: Adaptin ear-binding coat-associated protein 2 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 28.629941 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MEESEYESVL CVKPEVHVYR IPPRATNRGY RASEWQLDQP SWSGRLRITA KGKVAYIKLE DRTSGELFAQ APVDQFPGTA VESVTDSSR YFVIRIEDGN GRRAFIGLGF GDRGDAFDFN VALQDHFKWV KQQCEFAKQA QNPDEGPKLD LGFKDGQTIK I NIANMRKK ...String:
MEESEYESVL CVKPEVHVYR IPPRATNRGY RASEWQLDQP SWSGRLRITA KGKVAYIKLE DRTSGELFAQ APVDQFPGTA VESVTDSSR YFVIRIEDGN GRRAFIGLGF GDRGDAFDFN VALQDHFKWV KQQCEFAKQA QNPDEGPKLD LGFKDGQTIK I NIANMRKK EGAAGTPRAR PTSAGGLSLL PPPPGGKSST VIPPSGEQLS VGGSLVQPAV VSGSGGATEL WPQSKPAAAA TA DIWGDFT KSTGSPSSQS QPGTGWVQF

UniProtKB: Adaptin ear-binding coat-associated protein 2

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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

Concentration1.0 mg/mL
BufferpH: 8
GridModel: UltrAuFoil / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Details: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details: 4 UL OF SAMPLE/GRID WAS BLOTTED FOR 4 SECONDS AND PLUNGE FROZEN IN LIQUID-NITROGEN COOLED ETHANE.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
SoftwareName: Leginon
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number real images: 944 / Average electron dose: 1.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 36000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing #1

Image processing ID1
Startup modelType of model: OTHER
Details: Ab initio model generation performed in cryoSPARC v1
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 71571
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION

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Image processing #2

Image processing ID2
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 71571
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE

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Atomic model buiding 1

Initial model
PDB IDChain

2vgl

source_name: PDB, initial_model_type: experimental model

1tqz

source_name: PDB, initial_model_type: experimental model
DetailsSTARTING MODEL WAS GENERATED BY DOCKING PDB ENTRIES 2VGL AND 1TQZ INTO CRYO-EM DENSITY AND MANUALLY EDITING SEQUENCE AND STRUCTURAl CHANGES. MODEL REFINEMENT WAS PERFORMED USING ROSETTA AND PHENIX.REAL_SPACE_REFINE
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 63 / Target criteria: MAXIMUM LIKELIHOOD
Output model

PDB-6owo:
CRYO-EM STRUCTURE OF PHOSPHORYLATED AP-2 CORE BOUND TO NECAP

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