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- PDB-6rh6: Solution structure and 1H, 13C and 15N chemical shift assignments... -

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Basic information

Entry
Database: PDB / ID: 6rh6
TitleSolution structure and 1H, 13C and 15N chemical shift assignments for the complex of NECAP1 PHear domain with phosphorylated AP2 mu2 148-163
Components
  • AP-2 complex subunit mu
  • Adaptin ear-binding coat-associated protein 1
KeywordsENDOCYTOSIS / clathrin mediated endocytosis / regulation by phosphorylation / AP2 endocytic adaptor / NECAP / SNX9
Function / homology
Function and homology information


clathrin vesicle coat / Gap junction degradation / Formation of annular gap junctions / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / LDL clearance / Retrograde neurotrophin signalling / VLDLR internalisation and degradation / WNT5A-dependent internalization of FZD4 / extrinsic component of presynaptic endocytic zone membrane / MHC class II antigen presentation ...clathrin vesicle coat / Gap junction degradation / Formation of annular gap junctions / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / LDL clearance / Retrograde neurotrophin signalling / VLDLR internalisation and degradation / WNT5A-dependent internalization of FZD4 / extrinsic component of presynaptic endocytic zone membrane / MHC class II antigen presentation / AP-2 adaptor complex / regulation of vesicle size / postsynaptic neurotransmitter receptor internalization / Recycling pathway of L1 / positive regulation of synaptic vesicle endocytosis / Cargo recognition for clathrin-mediated endocytosis / clathrin adaptor activity / Clathrin-mediated endocytosis / vesicle budding from membrane / clathrin-dependent endocytosis / signal sequence binding / low-density lipoprotein particle receptor binding / Golgi Associated Vesicle Biogenesis / Trafficking of GluR2-containing AMPA receptors / positive regulation of receptor internalization / synaptic vesicle endocytosis / negative regulation of protein localization to plasma membrane / vesicle-mediated transport / clathrin-coated pit / intracellular protein transport / terminal bouton / receptor internalization / endocytosis / disordered domain specific binding / synaptic vesicle / protein transport / Cargo recognition for clathrin-mediated endocytosis / Clathrin-mediated endocytosis / protein-containing complex assembly / cytoplasmic vesicle / transmembrane transporter binding / postsynapse / synapse / lipid binding / glutamatergic synapse / plasma membrane / cytosol
Similarity search - Function
NECAP, PHear domain / Protein of unknown function (DUF1681) / Mu2, C-terminal domain / AP-2 complex subunit mu, N-terminal / Clathrin adaptor complexes medium chain signature 1. / Clathrin adaptor, mu subunit / Clathrin adaptor, mu subunit, conserved site / Clathrin adaptor complexes medium chain signature 2. / : / AP complex, mu/sigma subunit ...NECAP, PHear domain / Protein of unknown function (DUF1681) / Mu2, C-terminal domain / AP-2 complex subunit mu, N-terminal / Clathrin adaptor complexes medium chain signature 1. / Clathrin adaptor, mu subunit / Clathrin adaptor, mu subunit, conserved site / Clathrin adaptor complexes medium chain signature 2. / : / AP complex, mu/sigma subunit / Clathrin adaptor complex small chain / Adaptor complexes medium subunit family / AP-2 complex subunit mu, C-terminal superfamily / Mu homology domain / Mu homology domain (MHD) profile. / Longin-like domain superfamily / Pleckstrin-homology domain (PH domain)/Phosphotyrosine-binding domain (PTB) / PH-domain like / PH-like domain superfamily / Roll / Mainly Beta
Similarity search - Domain/homology
AP-2 complex subunit mu / Adaptin ear-binding coat-associated protein 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Rattus norvegicus (Norway rat)
MethodSOLUTION NMR / torsion angle dynamics / simulated annealing / molecular dynamics
AuthorsOwen, D.J. / Neuhaus, D. / Yang, J.-C. / Herrmann, T.
Funding support United Kingdom, Czech Republic, 5items
OrganizationGrant numberCountry
Wellcome Trust090909/Z/09/Z United Kingdom
Wellcome Trust097040/Z/11/Z United Kingdom
Medical Research Council (United Kingdom)U105178934 United Kingdom
European Communitys Seventh Framework ProgrammePIOF-GA-2012-330268
Czech Science FoundationGA18-05360S Czech Republic
CitationJournal: Dev.Cell / Year: 2019
Title: Temporal Ordering in Endocytic Clathrin-Coated Vesicle Formation via AP2 Phosphorylation.
Authors: Wrobel, A.G. / Kadlecova, Z. / Kamenicky, J. / Yang, J.C. / Herrmann, T. / Kelly, B.T. / McCoy, A.J. / Evans, P.R. / Martin, S. / Muller, S. / Sroubek, F. / Neuhaus, D. / Honing, S. / Owen, D.J.
History
DepositionApr 18, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 4, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Adaptin ear-binding coat-associated protein 1
B: AP-2 complex subunit mu


Theoretical massNumber of molelcules
Total (without water)17,3612
Polymers17,3612
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: isothermal titration calorimetry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area1210 Å2
ΔGint-6 kcal/mol
Surface area8940 Å2
MethodPISA
NMR ensembles
DataCriteria
Number of conformers (submitted / calculated)30 / 50structures with the lowest energy
RepresentativeModel #1lowest energy

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Components

#1: Protein Adaptin ear-binding coat-associated protein 1 / NECAP endocytosis-associated protein 1 / NECAP-1


Mass: 15562.371 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NECAP1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q8NC96
#2: Protein/peptide AP-2 complex subunit mu / AP-2 mu chain / Adaptor protein complex AP-2 subunit mu / Adaptor-related protein complex 2 subunit ...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: 1798.892 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Rattus norvegicus (Norway rat) / References: UniProt: P84092
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: SOLUTION NMR
NMR experiment
Conditions-IDExperiment-IDSolution-IDSample stateSpectrometer-IDType
122isotropic32D 1H-15N HSQC
132isotropic22D 1H-13C HSQC aliphatic
142isotropic22D 1H-13C HSQC aromatic
152isotropic32D 1H-1H NOESY (reject 15N,13C coupled 1H in F1, accept 15N,13C coupled 1H in F2) tau(m) 150ms
162isotropic32D 1H-1H NOESY (reject 15N,13C coupled 1H in F1 and F2) tau(m) 150ms
172isotropic32D 1H-1H TOCSY (reject 15N,13C coupled 1H in F2)
183isotropic32D 1H-13C HSQC aliphatic
193isotropic32D 1H-13C HSQC aromatic
1103isotropic32D 1H-13C HSQC aliphatic (constant time)
1113isotropic32D 1H-13C HSQC aromatic (constant time)
1123isotropic32D 1H-1H NOESY (reject 15N,13C coupled 1H in F1, accept 13C coupled 1H in F2) tau(m) 150ms
1132isotropic23D HBHA(CO)NH
1142isotropic23D HN(CA)CB
1153isotropic13D (H)CCH-COSY (1H,13C,1H)
1163isotropic13D (H)CCH-TOCSY (13C,13C,1H)
1172isotropic33D 1H-15N NOESY tau(m) 150ms
1182isotropic33D 1H-13C NOESY aliphatic tau(m) 150ms
1193isotropic33D 1H-13C NOESY aliphatic tau(m) 150ms
1202isotropic33D 1H-13C NOESY aromatic tau(m) 150ms
1213isotropic33D 1H-13C NOESY aromatic tau(m) 150ms
1222isotropic33D 1H-13C NOESY aliphatic (reject 15N,13C coupled 1H in F1) tau(m) 150ms
1233isotropic33D 1H-13C NOESY aliphatic (reject 15N,13C coupled 1H in F1) tau(m) 150ms
1242isotropic33D 1H-13C NOESY aromatic (reject 15N,13C coupled 1H in F1) tau(m) 150ms
1253isotropic33D 1H-13C NOESY aromatic (reject 15N,13C coupled 1H in F1) tau(m) 150ms

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Sample preparation

Details
TypeSolution-IDContentsLabelSolvent system
solution20.5 mM [U-98% 13C; U-98% 15N] NECAP1 1-133, 0.5 mM AP2 mu2 148-163, 70 mM [U-2H] sodium acetate, 95% H2O/5% D2O15N,13C_H2O_sample95% H2O/5% D2O
solution30.5 mM [U-98% 13C; U-98% 15N] NECAP1, 0.5 mM [U-98% 13C; U-98% 15N] AP2 mu2 148-163, 70 mM [U-2H] sodium acetate, 100% D2O15N,13C_D2O_sample100% D2O
Sample
Conc. (mg/ml)ComponentIsotopic labelingSolution-ID
0.5 mMNECAP1 1-133[U-98% 13C; U-98% 15N]2
0.5 mMAP2 mu2 148-163natural abundance2
70 mMsodium acetate[U-2H]2
0.5 mMNECAP1[U-98% 13C; U-98% 15N]3
0.5 mMAP2 mu2 148-163[U-98% 13C; U-98% 15N]3
70 mMsodium acetate[U-2H]3
Sample conditionsIonic strength: 70 mM / Label: conditions_1 / pH: 7 / Pressure: 1 atm / Temperature: 298 K

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NMR measurement

NMR spectrometer
TypeManufacturerModelField strength (MHz)Spectrometer-ID
Bruker AVANCE IIIBrukerAVANCE III6001
Bruker AVANCEBrukerAVANCE6002
Bruker AVANCE IIIBrukerAVANCE III8003

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Processing

NMR software
NameVersionDeveloperClassification
TopSpin3.5Bruker Biospinprocessing
CcpNmr Analysis2.4.2CCPNchemical shift assignment
Sparky3.115Goddardchemical shift assignment
UNIO2.8.1Herrmannstructure calculation
Xplor-NIH2.28Schwieters, Kuszewski, Tjandra and Clorestructure calculation
Amber11Case, Darden, Cheatham III, Simmerling, Wang, Duke, Luo, ... and Kollmanstructure calculation
Refinement
MethodSoftware ordinal
torsion angle dynamics3
simulated annealing4
molecular dynamics6
NMR representativeSelection criteria: lowest energy
NMR ensembleConformer selection criteria: structures with the lowest energy
Conformers calculated total number: 50 / Conformers submitted total number: 30

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