+データを開く
-基本情報
登録情報 | データベース: EMDB / ID: EMD-3385 | |||||||||
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タイトル | Cryo-EM structure of the human APC/C-Cdc20-Hsl1 complex | |||||||||
マップデータ | Single particle reconstruction of the human APC/C-Cdc20-Hsl1 complex | |||||||||
試料 |
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キーワード | Cell cycle / phosphorylation / mitosis / ubiquitination | |||||||||
機能・相同性 | 機能・相同性情報 protein localization to septin ring / mitotic morphogenesis checkpoint signaling / metaphase/anaphase transition of cell cycle / metaphase/anaphase transition of meiosis I / cellular bud neck septin ring / Inhibition of the proteolytic activity of APC/C required for the onset of anaphase by mitotic spindle checkpoint components / mitotic checkpoint complex / positive regulation of anaphase-promoting complex-dependent catabolic process / regulation of meiotic nuclear division / Conversion from APC/C:Cdc20 to APC/C:Cdh1 in late anaphase ...protein localization to septin ring / mitotic morphogenesis checkpoint signaling / metaphase/anaphase transition of cell cycle / metaphase/anaphase transition of meiosis I / cellular bud neck septin ring / Inhibition of the proteolytic activity of APC/C required for the onset of anaphase by mitotic spindle checkpoint components / mitotic checkpoint complex / positive regulation of anaphase-promoting complex-dependent catabolic process / regulation of meiotic nuclear division / 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 / Phosphorylation of Emi1 / 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 / anaphase-promoting complex binding / Phosphorylation of the APC/C / cellular bud neck / ubiquitin ligase activator activity / positive regulation of mitotic metaphase/anaphase transition / positive regulation of ubiquitin protein ligase activity / protein K11-linked ubiquitination / enzyme-substrate adaptor activity / regulation of mitotic metaphase/anaphase transition / positive regulation of dendrite morphogenesis / ubiquitin-ubiquitin ligase activity / mitotic sister chromatid cohesion / mitotic metaphase chromosome alignment / mitotic spindle assembly checkpoint signaling / cullin family protein binding / Regulation of APC/C activators between G1/S and early anaphase / ubiquitin-like ligase-substrate adaptor activity / Transcriptional Regulation by VENTX / mitotic spindle assembly / positive regulation of axon extension / heterochromatin / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / protein K48-linked ubiquitination / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Resolution of Sister Chromatid Cohesion / 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 / SCF-beta-TrCP mediated degradation of Emi1 / Assembly of the pre-replicative complex / RHO GTPases Activate Formins / Cdc20:Phospho-APC/C mediated degradation of Cyclin A / protein catabolic process / 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 pole / spindle / Separation of Sister Chromatids / ubiquitin-protein transferase activity / G2/M transition of mitotic cell cycle / microtubule cytoskeleton / ubiquitin protein ligase activity / Antigen processing: Ubiquitination & Proteasome degradation / nervous system development / mitotic cell cycle / Senescence-Associated Secretory Phenotype (SASP) / ubiquitin-dependent protein catabolic process / protein phosphatase binding / molecular adaptor activity / cell differentiation / non-specific serine/threonine protein kinase / regulation of cell cycle / Ub-specific processing proteases / protein kinase activity / protein ubiquitination / cell cycle / phosphorylation / cell division / negative regulation of gene expression / protein serine kinase activity / protein serine/threonine kinase activity / centrosome / ubiquitin protein ligase binding / nucleolus / zinc ion binding / nucleoplasm / ATP binding / nucleus / cytoplasm / cytosol 類似検索 - 分子機能 | |||||||||
生物種 | Homo sapiens (ヒト) / Saccharomyces cerevisiae (パン酵母) | |||||||||
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: 3.9 Å | |||||||||
データ登録者 | Zhang S / Chang L / Alfieri C / Zhang Z / Yang J / Maslen S / Skehel M / Barford D | |||||||||
引用 | ジャーナル: Nature / 年: 2016 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 |
ムービービューア |
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構造ビューア | EMマップ: SurfViewMolmilJmol/JSmol |
添付画像 |
-ダウンロードとリンク
-EMDBアーカイブ
マップデータ | emd_3385.map.gz | 10 MB | EMDBマップデータ形式 | |
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ヘッダ (付随情報) | emd-3385-v30.xml emd-3385.xml | 26.1 KB 26.1 KB | 表示 表示 | EMDBヘッダ |
画像 | EMD-3385.jpg | 76.9 KB | ||
アーカイブディレクトリ | http://ftp.pdbj.org/pub/emdb/structures/EMD-3385 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-3385 | HTTPS FTP |
-検証レポート
文書・要旨 | emd_3385_validation.pdf.gz | 234.8 KB | 表示 | EMDB検証レポート |
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文書・詳細版 | emd_3385_full_validation.pdf.gz | 233.9 KB | 表示 | |
XML形式データ | emd_3385_validation.xml.gz | 6.3 KB | 表示 | |
アーカイブディレクトリ | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3385 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3385 | HTTPS FTP |
-関連構造データ
-リンク
EMDBのページ | EMDB (EBI/PDBe) / EMDataResource |
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「今月の分子」の関連する項目 |
-マップ
ファイル | ダウンロード / ファイル: emd_3385.map.gz / 形式: CCP4 / 大きさ: 85.3 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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注釈 | Single particle reconstruction of the human APC/C-Cdc20-Hsl1 complex | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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
-試料の構成要素
+全体 : Recombinant human APC/C-Cdc20-Hsl1 complex
+超分子 #1000: Recombinant human APC/C-Cdc20-Hsl1 complex
+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
+分子 #15: Cell division cycle protein 20 homolog
+分子 #16: Hsl1
-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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解析 | 単粒子再構成法 |
試料の集合状態 | particle |
-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 |
-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 | 2015年10月30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: 11000 / 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: FIELD EMISSION GUN |
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 |
-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: 3.9 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: 179660 |