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- EMDB-20220: CRYO-EM STRUCTURE OF PHOSPHORYLATED AP-2 (mu E302K) BOUND TO NECA... -

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Basic information

Entry
Database: EMDB / ID: EMD-20220
TitleCRYO-EM STRUCTURE OF PHOSPHORYLATED AP-2 (mu E302K) BOUND TO NECAP IN THE PRESENCE OF SS DNA
Map data
SamplePhosphorylated AP2-NECAP (mu E302K) co-complex in the presence of ss DNA
  • (AP-2 complex subunit ...) x 4
  • Adaptin ear-binding coat-associated protein 2
  • Unknown region of Adaptin ear-binding coat-associated protein 2 Ex-domain
Function / homology
Function and homology information


Gap junction degradation / Formation of annular gap junctions / Retrograde neurotrophin signalling / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / Retrograde neurotrophin signalling / MHC class II antigen presentation / WNT5A-dependent internalization of FZD4 / LDL clearance / VLDLR internalisation and degradation ...Gap junction degradation / Formation of annular gap junctions / Retrograde neurotrophin signalling / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / Retrograde neurotrophin signalling / MHC class II antigen presentation / WNT5A-dependent internalization of FZD4 / LDL clearance / VLDLR internalisation and degradation / Clathrin-mediated endocytosis / Trafficking of GluR2-containing AMPA receptors / MHC class II antigen presentation / Cargo recognition for clathrin-mediated endocytosis / Cargo recognition for clathrin-mediated endocytosis / WNT5A-dependent internalization of FZD4 / Clathrin-mediated endocytosis / VLDLR internalisation and degradation / Recycling pathway of L1 / LDL clearance / Trafficking of GluR2-containing AMPA receptors / Recycling pathway of L1 / regulation of vesicle size / clathrin vesicle coat / clathrin adaptor complex / clathrin adaptor activity / cardiac septum development / neurotransmitter receptor internalization / vesicle budding from membrane / AP-2 adaptor complex / positive regulation of protein localization to membrane / postsynaptic neurotransmitter receptor internalization / coronary vasculature development / positive regulation of synaptic vesicle endocytosis / clathrin coat assembly / clathrin-dependent endocytosis / signal sequence binding / cargo receptor activity / negative regulation of protein localization to plasma membrane / aorta development / positive regulation of endocytosis / clathrin binding / ventricular septum development / positive regulation of receptor internalization / synaptic vesicle endocytosis / low-density lipoprotein particle receptor binding / presynapse / vesicle-mediated transport / receptor internalization / negative regulation of neuron death / secretory granule / intracellular protein transport / postsynapse / protein transport / protein-containing complex assembly / endocytosis / disordered domain specific binding / heart development / ion channel binding / lipid binding / protein-containing complex binding / intracellular membrane-bounded organelle / glutamatergic synapse / mitochondrion / plasma membrane
PH-like domain superfamily / Longin-like domain superfamily / Beta-adaptin appendage, C-terminal subdomain / Clathrin adaptor, appendage, Ig-like subdomain superfamily / Clathrin adaptor, beta-adaptin, appendage, Ig-like subdomain / NECAP, PHear domain / TBP domain superfamily / Armadillo-like helical / Coatomer/calthrin adaptor appendage, C-terminal subdomain / AP-1/2/4 complex subunit beta ...PH-like domain superfamily / Longin-like domain superfamily / Beta-adaptin appendage, C-terminal subdomain / Clathrin adaptor, appendage, Ig-like subdomain superfamily / Clathrin adaptor, beta-adaptin, appendage, Ig-like subdomain / NECAP, PHear domain / TBP domain superfamily / Armadillo-like helical / Coatomer/calthrin adaptor appendage, C-terminal subdomain / AP-1/2/4 complex subunit beta / Clathrin adaptor, alpha/beta/gamma-adaptin, appendage, Ig-like subdomain / Clathrin/coatomer adaptor, adaptin-like, N-terminal / Clathrin adaptor, mu subunit / Clathrin adaptor complex, small chain / Armadillo / Armadillo-type fold / Adaptor protein complex, sigma subunit / Adaptin N terminal region / Clathrin adaptor complexes medium chain signature 2. / Clathrin adaptor complexes medium chain signature 1. / Clathrin adaptor complexes small chain signature. / Beta2-adaptin appendage, C-terminal sub-domain / Protein of unknown function (DUF1681) / Adaptin C-terminal domain / Clathrin adaptor complex small chain / Clathrin adaptor, mu subunit, conserved site / Adaptor complexes medium subunit family / AP-2 complex subunit mu, C-terminal superfamily / Mu homology domain / AP-2 complex subunit sigma / AP complex subunit beta / AP complex, mu/sigma subunit / Mu homology domain (MHD) profile.
AP-2 complex subunit sigma / AP-2 complex subunit mu / Adaptin ear-binding coat-associated protein 2 / AP-2 complex subunit beta
Biological speciesMus musculus (house mouse) / Rattus norvegicus (Norway rat)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsPartlow EA / Baker RW / Beacham GM / Chappie J / Leschziner AE / Hollopeter G
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesR01 GM127548-01A1 United States
CitationJournal: Elife / Year: 2019
Title: A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex.
Authors: Edward A Partlow / Richard W Baker / Gwendolyn M Beacham / Joshua S Chappie / Andres E Leschziner / Gunther Hollopeter /
Abstract: Endocytosis of transmembrane proteins is orchestrated by the AP2 clathrin adaptor complex. AP2 dwells in a closed, inactive state in the cytosol, but adopts an open, active conformation on the plasma ...Endocytosis of transmembrane proteins is orchestrated by the AP2 clathrin adaptor complex. AP2 dwells in a closed, inactive state in the cytosol, but adopts an open, active conformation on the plasma membrane. Membrane-activated complexes are also phosphorylated, but the significance of this mark is debated. We recently proposed that NECAP negatively regulates AP2 by binding open and phosphorylated complexes (Beacham et al., 2018). Here, we report high-resolution cryo-EM structures of NECAP bound to phosphorylated AP2. The site of AP2 phosphorylation is directly coordinated by residues of the NECAP PHear domain that are predicted from genetic screens in . Using membrane mimetics to generate conformationally open AP2, we find that a second domain of NECAP binds these complexes and cryo-EM reveals both domains of NECAP engaging closed, inactive AP2. Assays in vitro and in vivo confirm these domains cooperate to inactivate AP2. We propose that phosphorylation marks adaptors for inactivation.
Validation ReportPDB-ID: 6oxl

SummaryFull reportAbout validation report
History
DepositionMay 13, 2019-
Header (metadata) releaseAug 28, 2019-
Map releaseSep 11, 2019-
UpdateSep 11, 2019-
Current statusSep 11, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.1
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1.1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6oxl
  • Surface level: 1.1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_20220.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.16 Å/pix.
x 300 pix.
= 348. Å
1.16 Å/pix.
x 300 pix.
= 348. Å
1.16 Å/pix.
x 300 pix.
= 348. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.16 Å
Density
Contour LevelBy AUTHOR: 1.1 / Movie #1: 1.1
Minimum - Maximum-0.45364538 - 1.9733219
Average (Standard dev.)0.0016241531 (±0.08705949)
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-3.5086.9290.002

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Supplemental data

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Sample components

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Entire Phosphorylated AP2-NECAP (mu E302K) co-complex in the presence of...

EntireName: Phosphorylated AP2-NECAP (mu E302K) co-complex in the presence of ss DNA
Number of components: 7

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Component #1: protein, Phosphorylated AP2-NECAP (mu E302K) co-complex in the pr...

ProteinName: Phosphorylated AP2-NECAP (mu E302K) co-complex in the presence of ss DNA
Recombinant expression: No
SourceSpecies: Mus musculus (house mouse)

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Component #2: protein, AP-2 complex subunit alpha-2

ProteinName: AP-2 complex subunit alpha-2 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 69.656297 kDa
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #3: protein, AP-2 complex subunit beta

ProteinName: AP-2 complex subunit beta / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 66.953195 kDa
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #4: protein, AP-2 complex subunit mu

ProteinName: AP-2 complex subunit mu / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 49.806688 kDa
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #5: protein, AP-2 complex subunit sigma

ProteinName: AP-2 complex subunit sigma / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 17.038688 kDa
SourceSpecies: Rattus norvegicus (Norway rat)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #6: protein, Adaptin ear-binding coat-associated protein 2

ProteinName: Adaptin ear-binding coat-associated protein 2 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 28.629941 kDa
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #7: protein, Unknown region of Adaptin ear-binding coat-associated pr...

ProteinName: Unknown region of Adaptin ear-binding coat-associated protein 2 Ex-domain
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.613749 kDa
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 1 mg/mL / pH: 8
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277.15 K / Humidity: 100 %
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 imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
ImagingMicroscope: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 1 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 36000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 600.0 - 2500.0 nm
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 324922
3D reconstructionSoftware: cryoSPARC / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Details: Non-Uniform refinement in cryoSPARC v2
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Refinement space: REAL
Details: STARTING 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
Input PDB model: 2VGL, 1TQZ
Output model

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