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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 data
SamplePHOSPHORYLATED AP2-NECAP CO-COMPLEX
  • (AP-2 complex subunit ...) x 4
  • Adaptin ear-binding coat-associated protein 2
Function / homology
Function and homology information


regulation of vesicle size / cardiac septum development / clathrin adaptor complex / cargo adaptor activity / clathrin vesicle coat / postsynaptic neurotransmitter receptor internalization / AP-2 adaptor complex / positive regulation of protein localization to membrane / clathrin adaptor activity / membrane coat ...regulation of vesicle size / cardiac septum development / clathrin adaptor complex / cargo adaptor activity / clathrin vesicle coat / postsynaptic neurotransmitter receptor internalization / AP-2 adaptor complex / positive regulation of protein localization to membrane / clathrin adaptor activity / membrane coat / coronary vasculature development / positive regulation of synaptic vesicle endocytosis / neurotransmitter receptor internalization / vesicle budding from membrane / signal sequence binding / clathrin coat assembly / clathrin-dependent endocytosis / aorta development / negative regulation of protein localization to plasma membrane / positive regulation of endocytosis / clathrin binding / ventricular septum development / synaptic vesicle endocytosis / positive regulation of receptor internalization / low-density lipoprotein particle receptor binding / regulation of hematopoietic stem cell differentiation / phosphatidylinositol binding / clathrin-coated pit / presynapse / vesicle-mediated transport / receptor internalization / negative regulation of neuron death / secretory granule / intracellular protein transport / kinase binding / postsynapse / protein transport / endocytosis / receptor-mediated endocytosis / kidney development / synaptic vesicle / protein-containing complex assembly / disordered domain specific binding / heart development / ion channel binding / cytoplasmic vesicle / lipid binding / glutamatergic synapse / intracellular membrane-bounded organelle / protein domain specific binding / protein-containing complex binding / protein kinase binding / mitochondrion / plasma membrane
NECAP, PHear domain / Clathrin adaptor, mu subunit / Adaptor protein complex, sigma subunit / Clathrin adaptor, beta-adaptin, appendage, Ig-like subdomain / Armadillo-type fold / Beta-adaptin appendage, C-terminal subdomain / Clathrin adaptor, appendage, Ig-like subdomain superfamily / Armadillo / Clathrin adaptor complex, small chain / Clathrin/coatomer adaptor, adaptin-like, N-terminal ...NECAP, PHear domain / Clathrin adaptor, mu subunit / Adaptor protein complex, sigma subunit / Clathrin adaptor, beta-adaptin, appendage, Ig-like subdomain / Armadillo-type fold / Beta-adaptin appendage, C-terminal subdomain / Clathrin adaptor, appendage, Ig-like subdomain superfamily / Armadillo / Clathrin adaptor complex, small chain / Clathrin/coatomer adaptor, adaptin-like, N-terminal / Clathrin adaptor, mu subunit, conserved site / Clathrin adaptor, alpha-adaptin, appendage, C-terminal subdomain / Clathrin adaptor, alpha/beta/gamma-adaptin, appendage, Ig-like subdomain / Coatomer/calthrin adaptor appendage, C-terminal subdomain / Longin-like domain superfamily / Armadillo-like helical / PH-like domain superfamily / TBP domain superfamily / Adaptor protein complex AP-2, alpha subunit / AP-1/2/4 complex subunit beta / AP-2 complex subunit mu, C-terminal superfamily / AP-2 complex subunit sigma / AP complex, mu/sigma subunit / Mu homology domain / AP complex subunit beta / AP-2 complex subunit mu, N-terminal / Mu2, C-terminal domain
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
Biological speciesMus musculus (house mouse) / Rattus norvegicus (Norway rat)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsPartlow EA / Baker RW / Beacham GM / Chappie JS / Leschziner AE / 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: 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: 6owo

SummaryFull reportAbout validation report
History
DepositionMay 10, 2019-
Header (metadata) releaseAug 28, 2019-
Map releaseSep 11, 2019-
UpdateJan 8, 2020-
Current statusJan 8, 2020Processing 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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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

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)
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: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.09064991 - 0.174634
Average (Standard dev.)0.00004023629 (±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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Additional map: cryo-EM structure of phosphorylated AP2 bound to NECAP;...

Fileemd_20215_additional.map
Annotationcryo-EM structure of phosphorylated AP2 bound to NECAP; unsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: cryo-EM structure of phosphorylated AP2 bound to NECAP;...

Fileemd_20215_half_map_1.map
Annotationcryo-EM structure of phosphorylated AP2 bound to NECAP; unfiltered half-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: cryo-EM structure of phosphorylated AP2 bound to NECAP;...

Fileemd_20215_half_map_2.map
Annotationcryo-EM structure of phosphorylated AP2 bound to NECAP; unfiltered half-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire PHOSPHORYLATED AP2-NECAP CO-COMPLEX

EntireName: PHOSPHORYLATED AP2-NECAP CO-COMPLEX / Number of components: 6

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Component #1: protein, PHOSPHORYLATED AP2-NECAP CO-COMPLEX

ProteinName: PHOSPHORYLATED AP2-NECAP CO-COMPLEX / 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: 49.806621 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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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 1 mg/mL / pH: 8
Support filmGLOW DISCHARGE
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 acquisition

Image acquisitionNumber of digital images: 944

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 71571
3D reconstruction #1Software: RELION / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF
3D reconstruction #2Software: RELION / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Target criteria: MAXIMUM LIKELIHOOD / 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 ...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
Overall bvalue: 63
Output model

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