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データを開く
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基本情報
登録情報 | データベース: EMDB / ID: EMD-3386 | |||||||||
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タイトル | Cryo-EM structure of human apo unphosphorylated APC/C | |||||||||
![]() | Single particle reconstruction of human apo unphosphorylated APC/C | |||||||||
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![]() | Cell cycle / phosphorylation / mitosis / ubiquitination | |||||||||
機能・相同性 | ![]() Conversion from APC/C:Cdc20 to APC/C:Cdh1 in late anaphase / regulation of mitotic cell cycle spindle assembly checkpoint / positive regulation of synapse maturation / Inactivation of APC/C via direct inhibition of the APC/C complex / APC/C:Cdc20 mediated degradation of mitotic proteins / anaphase-promoting complex / Aberrant regulation of mitotic exit in cancer due to RB1 defects / regulation of meiotic cell cycle / metaphase/anaphase transition of mitotic cell cycle / anaphase-promoting complex-dependent catabolic process ...Conversion from APC/C:Cdc20 to APC/C:Cdh1 in late anaphase / regulation of mitotic cell cycle spindle assembly checkpoint / positive regulation of synapse maturation / Inactivation of APC/C via direct inhibition of the APC/C complex / APC/C:Cdc20 mediated degradation of mitotic proteins / anaphase-promoting complex / Aberrant regulation of mitotic exit in cancer due to RB1 defects / regulation of meiotic cell cycle / metaphase/anaphase transition of mitotic cell cycle / anaphase-promoting complex-dependent catabolic process / positive regulation of synaptic plasticity / regulation of exit from mitosis / Phosphorylation of the APC/C / positive regulation of mitotic metaphase/anaphase transition / protein K11-linked ubiquitination / enzyme-substrate adaptor activity / positive regulation of dendrite morphogenesis / regulation of mitotic metaphase/anaphase transition / ubiquitin-ubiquitin ligase activity / mitotic metaphase chromosome alignment / cullin family protein binding / Regulation of APC/C activators between G1/S and early anaphase / Transcriptional Regulation by VENTX / positive regulation of axon extension / protein K48-linked ubiquitination / heterochromatin / regulation of mitotic cell cycle / APC/C:Cdc20 mediated degradation of Cyclin B / APC-Cdc20 mediated degradation of Nek2A / nuclear periphery / Autodegradation of Cdh1 by Cdh1:APC/C / APC/C:Cdc20 mediated degradation of Securin / Assembly of the pre-replicative complex / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / APC/C:Cdh1 mediated degradation of Cdc20 and other APC/C:Cdh1 targeted proteins in late mitosis/early G1 / brain development / CDK-mediated phosphorylation and removal of Cdc6 / mitotic spindle / kinetochore / spindle / Separation of Sister Chromatids / ubiquitin-protein transferase activity / microtubule cytoskeleton / ubiquitin protein ligase activity / Antigen processing: Ubiquitination & Proteasome degradation / mitotic cell cycle / nervous system development / Senescence-Associated Secretory Phenotype (SASP) / ubiquitin-dependent protein catabolic process / protein phosphatase binding / molecular adaptor activity / cell differentiation / protein ubiquitination / cell division / negative regulation of gene expression / centrosome / ubiquitin protein ligase binding / nucleolus / zinc ion binding / nucleoplasm / nucleus / cytosol / cytoplasm 類似検索 - 分子機能 | |||||||||
生物種 | ![]() | |||||||||
手法 | 単粒子再構成法 / クライオ電子顕微鏡法 / ネガティブ染色法 / 解像度: 3.8 Å | |||||||||
![]() | Zhang S / Chang L / Alfieri C / Zhang Z / Yang J / Maslen S / Skehel M / Barford D | |||||||||
![]() | ![]() タイトル: Molecular mechanism of APC/C activation by mitotic phosphorylation. 著者: Suyang Zhang / Leifu Chang / Claudio Alfieri / Ziguo Zhang / Jing Yang / Sarah Maslen / Mark Skehel / David Barford / ![]() 要旨: In eukaryotes, the anaphase-promoting complex (APC/C, also known as the cyclosome) regulates the ubiquitin-dependent proteolysis of specific cell-cycle proteins to coordinate chromosome segregation ...In eukaryotes, the anaphase-promoting complex (APC/C, also known as the cyclosome) regulates the ubiquitin-dependent proteolysis of specific cell-cycle proteins to coordinate chromosome segregation in mitosis and entry into the G1 phase. The catalytic activity of the APC/C and its ability to specify the destruction of particular proteins at different phases of the cell cycle are controlled by its interaction with two structurally related coactivator subunits, Cdc20 and Cdh1. Coactivators recognize substrate degrons, and enhance the affinity of the APC/C for its cognate E2 (refs 4-6). During mitosis, cyclin-dependent kinase (Cdk) and polo-like kinase (Plk) control Cdc20- and Cdh1-mediated activation of the APC/C. Hyperphosphorylation of APC/C subunits, notably Apc1 and Apc3, is required for Cdc20 to activate the APC/C, whereas phosphorylation of Cdh1 prevents its association with the APC/C. Since both coactivators associate with the APC/C through their common C-box and Ile-Arg tail motifs, the mechanism underlying this differential regulation is unclear, as is the role of specific APC/C phosphorylation sites. Here, using cryo-electron microscopy and biochemical analysis, we define the molecular basis of how phosphorylation of human APC/C allows for its control by Cdc20. An auto-inhibitory segment of Apc1 acts as a molecular switch that in apo unphosphorylated APC/C interacts with the C-box binding site and obstructs engagement of Cdc20. Phosphorylation of the auto-inhibitory segment displaces it from the C-box-binding site. Efficient phosphorylation of the auto-inhibitory segment, and thus relief of auto-inhibition, requires the recruitment of Cdk-cyclin in complex with a Cdk regulatory subunit (Cks) to a hyperphosphorylated loop of Apc3. We also find that the small-molecule inhibitor, tosyl-l-arginine methyl ester, preferentially suppresses APC/C(Cdc20) rather than APC/C(Cdh1), and interacts with the binding sites of both the C-box and Ile-Arg tail motifs. Our results reveal the mechanism for the regulation of mitotic APC/C by phosphorylation and provide a rationale for the development of selective inhibitors of this state. | |||||||||
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 | EMマップ: ![]() ![]() ![]() |
添付画像 |
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ダウンロードとリンク
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マップデータ | ![]() | 8.7 MB | ![]() | |
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ヘッダ (付随情報) | ![]() ![]() | 23.7 KB 23.7 KB | 表示 表示 | ![]() |
画像 | ![]() | 81.7 KB | ||
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
EMDBのページ | ![]() ![]() |
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「今月の分子」の関連する項目 |
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マップ
ファイル | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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注釈 | Single particle reconstruction of human apo unphosphorylated APC/C | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ボクセルのサイズ | X=Y=Z: 1.36 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
密度 |
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対称性 | 空間群: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
詳細 | EMDB XML:
CCP4マップ ヘッダ情報:
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-添付データ
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試料の構成要素
+全体 : Recombinant human apo unphosphorylated APC/C
+超分子 #1000: Recombinant human apo unphosphorylated APC/C
+分子 #1: Anaphase-promoting complex subunit 1
+分子 #2: Anaphase-promoting complex subunit 2
+分子 #3: Anaphase-promoting complex subunit 3
+分子 #4: Anaphase-promoting complex subunit 4
+分子 #5: Anaphase-promoting complex subunit 5
+分子 #6: Anaphase-promoting complex subunit 6
+分子 #7: Anaphase-promoting complex subunit 7
+分子 #8: Anaphase-promoting complex subunit 8
+分子 #9: Anaphase-promoting complex subunit 10
+分子 #10: Anaphase-promoting complex subunit 11
+分子 #11: Anaphase-promoting complex subunit 12
+分子 #12: Anaphase-promoting complex subunit 13
+分子 #13: Anaphase-promoting complex subunit 15
+分子 #14: Anaphase-promoting complex subunit 16
-実験情報
-構造解析
手法 | ネガティブ染色法, クライオ電子顕微鏡法 |
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![]() | 単粒子再構成法 |
試料の集合状態 | particle |
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試料調製
濃度 | 0.15 mg/mL |
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緩衝液 | pH: 8 / 詳細: 20mM Hepes, 150mM NaCl, 0.005mM TCEP |
染色 | タイプ: NEGATIVE / 詳細: Vitrification in liquid ethane |
グリッド | 詳細: 200 mesh Quantifoil R2/2 copper grid with thin carbon support, treated with a 9:1 argon:oxygen plasma cleaner for 20-40s |
凍結 | 凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 100 K / 装置: FEI VITROBOT MARK III 手法: The grids were incubated for 30s at 4 degree and 100% humidity before blotting for 5s and plunging into liquid ethane |
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電子顕微鏡法
顕微鏡 | FEI POLARA 300 |
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温度 | 最低: 90 K / 最高: 120 K / 平均: 105 K |
特殊光学系 | エネルギーフィルター - 名称: FEI |
日付 | 2014年11月1日 |
撮影 | カテゴリ: CCD フィルム・検出器のモデル: FEI FALCON II (4k x 4k) 実像数: 4500 / 平均電子線量: 27 e/Å2 詳細: The exposure time for each micrograph was 2s at a dose rate of 27 electrons/angstrom2/s. 34 movie frames were recorded for each micrograph. |
電子線 | 加速電圧: 300 kV / 電子線源: ![]() |
電子光学系 | 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / Cs: 2 mm / 最大 デフォーカス(公称値): 4.0 µm / 最小 デフォーカス(公称値): 2.0 µm / 倍率(公称値): 78000 |
試料ステージ | 試料ホルダー: liquid nitrogen cooled / 試料ホルダーモデル: OTHER |
実験機器 | ![]() モデル: Tecnai Polara / 画像提供: FEI Company |
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画像解析
詳細 | Particles were selected by automatic particle picking in RELION |
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CTF補正 | 詳細: Each micrograph |
最終 再構成 | 想定した対称性 - 点群: C1 (非対称) / 解像度のタイプ: BY AUTHOR / 解像度: 3.8 Å / 解像度の算出法: OTHER / ソフトウェア - 名称: RELION / 使用した粒子像数: 347317 |