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- PDB-6oxl: CRYO-EM STRUCTURE OF PHOSPHORYLATED AP-2 (mu E302K) BOUND TO NECA... -

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Basic information

Entry
Database: PDB / ID: 6oxl
TitleCRYO-EM STRUCTURE OF PHOSPHORYLATED AP-2 (mu E302K) BOUND TO NECAP IN THE PRESENCE OF SS DNA
Components
  • (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
KeywordsENDOCYTOSIS / AP2 / NECAP2 PROTEIN / CYTOPLASMIC VESICLE / LIPID-BINDING / ADAPTOR / MEMBRANE / TRANSPORT / PHOSPHORYLATION
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 / cargo receptor activity / signal sequence binding / clathrin-dependent endocytosis / negative regulation of protein localization to plasma membrane / aorta development / clathrin binding / positive regulation of endocytosis / ventricular septum development / positive regulation of receptor internalization / low-density lipoprotein particle receptor binding / synaptic vesicle endocytosis / presynapse / vesicle-mediated transport / receptor internalization / secretory granule / negative regulation of neuron death / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsPartlow, E.A. / Baker, R.W. / Beacham, G.M. / Chappie, J. / Leschziner, A.E. / Hollopeter, G.
Funding support United States, 1items
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 Report
SummaryFull reportAbout validation report
History
DepositionMay 13, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 11, 2019Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: AP-2 complex subunit alpha-2
B: AP-2 complex subunit beta
M: AP-2 complex subunit mu
S: AP-2 complex subunit sigma
N: Adaptin ear-binding coat-associated protein 2
n: Unknown region of Adaptin ear-binding coat-associated protein 2 Ex-domain


Theoretical massNumber of molelcules
Total (without water)232,6996
Polymers232,6996
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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AP-2 complex subunit ... , 4 types, 4 molecules ABMS

#1: Protein/peptide AP-2 complex subunit alpha-2


Mass: 69656.297 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ap2a2, Adtab / Plasmid: PACYC_DUET / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Protein/peptide AP-2 complex subunit beta / AP105B / Adaptor protein complex AP-2 subunit beta / Adaptor-related protein complex 2 subunit beta / Beta-2-adaptin / Beta-adaptin / Clathrin assembly protein complex 2 beta large chain / Plasma membrane adaptor HA2/AP2 adaptin beta subunit


Mass: 66953.195 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ap2b1, Clapb1 / Plasmid: PET_DUET / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9DBG3
#3: Protein/peptide AP-2 complex subunit mu / AP-2 mu chain / Adaptor protein complex AP-2 subunit mu / Adaptor-related protein complex 2 subunit mu / Clathrin assembly protein complex 2 mu medium chain / Clathrin coat assembly protein AP50 / Clathrin coat-associated protein AP50 / Mu2-adaptin / Plasma membrane adaptor AP-2 50 kDa protein


Mass: 49806.688 Da / Num. of mol.: 1 / Mutation: E302K
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ap2m1, Clapm1 / Plasmid: PET_DUET / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P84091
#4: Protein/peptide AP-2 complex subunit sigma / Adaptor protein complex AP-2 subunit sigma / Adaptor-related protein complex 2 subunit sigma / Clathrin assembly protein 2 sigma small chain / Clathrin coat assembly protein AP17 / Clathrin coat-associated protein AP17 / Plasma membrane adaptor AP-2 17 kDa protein / Sigma-adaptin 3b / Sigma2-adaptin


Mass: 17038.688 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Ap2s1, Ap17, Claps2 / Plasmid: PACYC_DUET / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P62744

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Protein/peptide , 2 types, 2 molecules Nn

#5: Protein/peptide Adaptin ear-binding coat-associated protein 2 / NECAP endocytosis-associated protein 2 / NECAP-2


Mass: 28629.941 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Necap2 / Plasmid: PET-21B / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9D1J1
#6: Protein/peptide Unknown region of Adaptin ear-binding coat-associated protein 2 Ex-domain


Mass: 613.749 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Plasmid: PET21-B / Production host: Escherichia coli BL21(DE3) (bacteria)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Phosphorylated AP2-NECAP (mu E302K) co-complex in the presence of ss DNA
Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5, 6 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
SpecimenConc.: 1 mg/ml
Details: Contains an E302K mutation in the mu subunit of AP2.
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
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
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 36000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 1 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameCategory
1RELIONparticle selection
2Leginonimage acquisition
4CTFFINDCTF correction
5GctfCTF correction
6RELIONCTF correction
9UCSF Chimeramodel fitting
11RELIONinitial Euler assignment
12cryoSPARCfinal Euler assignment
13cryoSPARCclassification
14cryoSPARC3D reconstruction
15Rosettamodel refinement
16PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 324922 / Details: Non-Uniform refinement in cryoSPARC v2 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / 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
Atomic model building
IDPDB-ID3D fitting-ID
12VGL1
21TQZ1

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