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基本情報
登録情報 | データベース: PDB / ID: 6oxl | ||||||
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タイトル | CRYO-EM STRUCTURE OF PHOSPHORYLATED AP-2 (mu E302K) BOUND TO NECAP IN THE PRESENCE OF SS DNA | ||||||
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![]() | ENDOCYTOSIS / AP2 / NECAP2 PROTEIN / CYTOPLASMIC VESICLE / LIPID-BINDING / ADAPTOR / MEMBRANE / TRANSPORT / PHOSPHORYLATION | ||||||
機能・相同性 | ![]() 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 / AP-2 adaptor complex / regulation of vesicle size / 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 adaptor activity / Clathrin-mediated endocytosis / vesicle budding from membrane / clathrin-dependent endocytosis / MHC class II antigen presentation / signal sequence binding / coronary vasculature development / regulation of hematopoietic stem cell differentiation / aorta development / ventricular septum development / clathrin binding / low-density lipoprotein particle receptor binding / 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 / Neutrophil degranulation / secretory granule / kidney development / intracellular protein transport / cytoplasmic side of plasma membrane / kinase binding / 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 / synapse / lipid binding / protein kinase binding / glutamatergic synapse / mitochondrion / plasma membrane 類似検索 - 分子機能 | ||||||
生物種 | ![]() ![]() ![]() ![]() | ||||||
手法 | 電子顕微鏡法 / 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 3.5 Å | ||||||
![]() | Partlow, E.A. / Baker, R.W. / Beacham, G.M. / Chappie, J. / Leschziner, A.E. / Hollopeter, G. | ||||||
資金援助 | ![]()
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![]() | ![]() タイトル: A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex. 著者: Edward A Partlow / Richard W Baker / Gwendolyn M Beacham / Joshua S Chappie / Andres E Leschziner / Gunther Hollopeter / ![]() 要旨: 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. | ||||||
履歴 |
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構造の表示
ムービー |
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構造ビューア | 分子: ![]() ![]() |
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PDBx/mmCIF形式 | ![]() | 366.4 KB | 表示 | ![]() |
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PDB形式 | ![]() | 291.6 KB | 表示 | ![]() |
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-検証レポート
文書・要旨 | ![]() | 1.3 MB | 表示 | ![]() |
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文書・詳細版 | ![]() | 1.3 MB | 表示 | |
XML形式データ | ![]() | 61.9 KB | 表示 | |
CIF形式データ | ![]() | 93.4 KB | 表示 | |
アーカイブディレクトリ | ![]() ![]() | HTTPS FTP |
-関連構造データ
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リンク
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集合体
登録構造単位 | ![]()
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要素
-AP-2 complex subunit ... , 4種, 4分子 ABMS
#1: タンパク質 | 分子量: 69656.297 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) ![]() ![]() ![]() ![]() |
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#2: タンパク質 | 分子量: 66953.195 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) ![]() ![]() ![]() ![]() |
#3: タンパク質 | 分子量: 49806.688 Da / 分子数: 1 / Mutation: E302K / 由来タイプ: 組換発現 / 由来: (組換発現) ![]() ![]() ![]() ![]() |
#4: タンパク質 | 分子量: 17038.688 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) ![]() ![]() ![]() ![]() |
-タンパク質 / タンパク質・ペプチド , 2種, 2分子 Nn
#5: タンパク質 | 分子量: 28629.941 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) ![]() ![]() ![]() ![]() |
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#6: タンパク質・ペプチド | 分子量: 613.749 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) ![]() ![]() ![]() ![]() |
-詳細
Has protein modification | Y |
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-実験情報
-実験
実験 | 手法: 電子顕微鏡法 |
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EM実験 | 試料の集合状態: PARTICLE / 3次元再構成法: 単粒子再構成法 |
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試料調製
構成要素 | 名称: Phosphorylated AP2-NECAP (mu E302K) co-complex in the presence of ss DNA タイプ: COMPLEX / Entity ID: all / 由来: MULTIPLE SOURCES |
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分子量 | 実験値: NO |
由来(天然) | 生物種: ![]() ![]() |
由来(組換発現) | 生物種: ![]() ![]() |
緩衝液 | pH: 8 |
試料 | 濃度: 1 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES 詳細: Contains an E302K mutation in the mu subunit of AP2. |
試料支持 | グリッドの材料: GOLD / グリッドのサイズ: 300 divisions/in. / グリッドのタイプ: UltrAuFoil |
急速凍結 | 装置: FEI VITROBOT MARK IV / 凍結剤: ETHANE / 湿度: 100 % / 凍結前の試料温度: 277.15 K 詳細: 4 UL OF SAMPLE/GRID WAS BLOTTED FOR 4 SECONDS AND PLUNGE FROZEN IN LIQUID-NITROGEN COOLED ETHANE |
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電子顕微鏡撮影
実験機器 | ![]() モデル: Talos Arctica / 画像提供: FEI Company |
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顕微鏡 | モデル: FEI TALOS ARCTICA |
電子銃 | 電子線源: ![]() |
電子レンズ | モード: BRIGHT FIELD / 倍率(公称値): 36000 X / 最大 デフォーカス(公称値): 2500 nm / 最小 デフォーカス(公称値): 600 nm / Cs: 2.7 mm |
試料ホルダ | 凍結剤: NITROGEN |
撮影 | 電子線照射量: 1 e/Å2 / 検出モード: COUNTING フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) |
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解析
EMソフトウェア |
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CTF補正 | タイプ: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||||||||||||||||
対称性 | 点対称性: C1 (非対称) | |||||||||||||||||||||||||||||||||||||||
3次元再構成 | 解像度: 3.5 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 324922 / 詳細: Non-Uniform refinement in cryoSPARC v2 / 対称性のタイプ: POINT | |||||||||||||||||||||||||||||||||||||||
原子モデル構築 | プロトコル: RIGID BODY FIT / 空間: REAL 詳細: 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 ...詳細: 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 | |||||||||||||||||||||||||||||||||||||||
原子モデル構築 |
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