+データを開く
-基本情報
登録情報 | データベース: PDB / ID: 2n1f | ||||||
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タイトル | Structure and assembly of the mouse ASC filament by combined NMR spectroscopy and cryo-electron microscopy | ||||||
要素 | Apoptosis-associated speck-like protein | ||||||
キーワード | APOPTOSIS (アポトーシス) / mouse ASC filament / ASC Apoptosis-associated speck like protein containing a CARD / PYRIN domain / inflammasomes (インフラマソーム) / death domain | ||||||
機能・相同性 | 機能・相同性情報 interleukin-1 beta production / CLEC7A/inflammasome pathway / positive regulation of chemokine production => GO:0032722 / Pyrin domain binding / myosin I binding / positive regulation of antigen processing and presentation of peptide antigen via MHC class II / regulation of cysteine-type endopeptidase activity involved in apoptotic process / myeloid dendritic cell activation involved in immune response / regulation of intrinsic apoptotic signaling pathway / peptidase activator activity involved in apoptotic process ...interleukin-1 beta production / CLEC7A/inflammasome pathway / positive regulation of chemokine production => GO:0032722 / Pyrin domain binding / myosin I binding / positive regulation of antigen processing and presentation of peptide antigen via MHC class II / regulation of cysteine-type endopeptidase activity involved in apoptotic process / myeloid dendritic cell activation involved in immune response / regulation of intrinsic apoptotic signaling pathway / peptidase activator activity involved in apoptotic process / myeloid dendritic cell activation / IkappaB kinase complex / AIM2 inflammasome complex / 飲作用 / NLRP1 inflammasome complex / interleukin-6 receptor binding / positive regulation of adaptive immune response / NLRP3 inflammasome complex / BMP receptor binding / negative regulation of protein serine/threonine kinase activity / negative regulation of interferon-beta production / positive regulation of cysteine-type endopeptidase activity / activation of cysteine-type endopeptidase activity / regulation of tumor necrosis factor-mediated signaling pathway / positive regulation of extrinsic apoptotic signaling pathway / cysteine-type endopeptidase activity involved in apoptotic process / tropomyosin binding / positive regulation of actin filament polymerization / regulation of GTPase activity / negative regulation of NF-kappaB transcription factor activity / positive regulation of activated T cell proliferation / positive regulation of release of cytochrome c from mitochondria / positive regulation of cysteine-type endopeptidase activity involved in apoptotic process / positive regulation of interleukin-10 production / intrinsic apoptotic signaling pathway by p53 class mediator / intrinsic apoptotic signaling pathway in response to DNA damage by p53 class mediator / negative regulation of cytokine production involved in inflammatory response / positive regulation of T cell migration / cellular response to interleukin-1 / positive regulation of phagocytosis / negative regulation of canonical NF-kappaB signal transduction / positive regulation of defense response to virus by host / tumor necrosis factor-mediated signaling pathway / activation of innate immune response / Neutrophil degranulation / positive regulation of interleukin-1 beta production / regulation of autophagy / positive regulation of interleukin-8 production / response to bacterium / positive regulation of JNK cascade / regulation of protein stability / protein homooligomerization / positive regulation of interleukin-6 production / activation of cysteine-type endopeptidase activity involved in apoptotic process / positive regulation of DNA-binding transcription factor activity / positive regulation of T cell activation / positive regulation of type II interferon production / positive regulation of tumor necrosis factor production / cellular response to tumor necrosis factor / positive regulation of NF-kappaB transcription factor activity / regulation of inflammatory response / defense response to virus / regulation of apoptotic process / cellular response to lipopolysaccharide / protease binding / defense response to Gram-negative bacterium / transmembrane transporter binding / positive regulation of ERK1 and ERK2 cascade / protein dimerization activity / 炎症 / positive regulation of apoptotic process / ゴルジ体 / 自然免疫系 / neuronal cell body / apoptotic process / 核小体 / enzyme binding / 小胞体 / protein homodimerization activity / protein-containing complex / ミトコンドリア / extracellular region / 核質 / identical protein binding / 細胞核 / 細胞質基質 / 細胞質 類似検索 - 分子機能 | ||||||
生物種 | Mus musculus (ハツカネズミ) | ||||||
手法 | 個体NMR / 電子顕微鏡法 / らせん対称体再構成法 / simulated annealing / クライオ電子顕微鏡法 / 解像度: 4 Å | ||||||
Model details | lowest energy, model1 | ||||||
データ登録者 | Sborgi, L. / Ravotti, F. / Dandey, V. / Dick, M. / Mazur, A. / Reckel, S. / Chami, M. / Scherer, S. / Bockmann, A. / Egelman, E. ...Sborgi, L. / Ravotti, F. / Dandey, V. / Dick, M. / Mazur, A. / Reckel, S. / Chami, M. / Scherer, S. / Bockmann, A. / Egelman, E. / Stahlberg, H. / Broz, P. / Meier, B. / Hiller, S. | ||||||
引用 | ジャーナル: Proc Natl Acad Sci U S A / 年: 2015 タイトル: Structure and assembly of the mouse ASC inflammasome by combined NMR spectroscopy and cryo-electron microscopy. 著者: Lorenzo Sborgi / Francesco Ravotti / Venkata P Dandey / Mathias S Dick / Adam Mazur / Sina Reckel / Mohamed Chami / Sebastian Scherer / Matthias Huber / Anja Böckmann / Edward H Egelman / ...著者: Lorenzo Sborgi / Francesco Ravotti / Venkata P Dandey / Mathias S Dick / Adam Mazur / Sina Reckel / Mohamed Chami / Sebastian Scherer / Matthias Huber / Anja Böckmann / Edward H Egelman / Henning Stahlberg / Petr Broz / Beat H Meier / Sebastian Hiller / 要旨: Inflammasomes are multiprotein complexes that control the innate immune response by activating caspase-1, thus promoting the secretion of cytokines in response to invading pathogens and endogenous ...Inflammasomes are multiprotein complexes that control the innate immune response by activating caspase-1, thus promoting the secretion of cytokines in response to invading pathogens and endogenous triggers. Assembly of inflammasomes is induced by activation of a receptor protein. Many inflammasome receptors require the adapter protein ASC [apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)], which consists of two domains, the N-terminal pyrin domain (PYD) and the C-terminal CARD. Upon activation, ASC forms large oligomeric filaments, which facilitate procaspase-1 recruitment. Here, we characterize the structure and filament formation of mouse ASC in vitro at atomic resolution. Information from cryo-electron microscopy and solid-state NMR spectroscopy is combined in a single structure calculation to obtain the atomic-resolution structure of the ASC filament. Perturbations of NMR resonances upon filament formation monitor the specific binding interfaces of ASC-PYD association. Importantly, NMR experiments show the rigidity of the PYD forming the core of the filament as well as the high mobility of the CARD relative to this core. The findings are validated by structure-based mutagenesis experiments in cultured macrophages. The 3D structure of the mouse ASC-PYD filament is highly similar to the recently determined human ASC-PYD filament, suggesting evolutionary conservation of ASC-dependent inflammasome mechanisms. | ||||||
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 | 分子: MolmilJmol/JSmol |
-ダウンロードとリンク
-ダウンロード
PDBx/mmCIF形式 | 2n1f.cif.gz | 2.1 MB | 表示 | PDBx/mmCIF形式 |
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PDB形式 | pdb2n1f.ent.gz | 1.9 MB | 表示 | PDB形式 |
PDBx/mmJSON形式 | 2n1f.json.gz | ツリー表示 | PDBx/mmJSON形式 | |
その他 | その他のダウンロード |
-検証レポート
アーカイブディレクトリ | https://data.pdbj.org/pub/pdb/validation_reports/n1/2n1f ftp://data.pdbj.org/pub/pdb/validation_reports/n1/2n1f | HTTPS FTP |
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-関連構造データ
-リンク
-集合体
登録構造単位 |
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1 |
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NMR アンサンブル |
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対称性 | らせん対称: (回転対称性: 3 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 8 / Rise per n subunits: 14.2 Å / Rotation per n subunits: 53 °) | |||||||||
詳細 | The helical parameters generate the filament from any single chain. |
-要素
#1: タンパク質 | 分子量: 10118.684 Da / 分子数: 15 / 断片: pyrin domain (UNP residues 2-90) / 由来タイプ: 組換発現 / 由来: (組換発現) Mus musculus (ハツカネズミ) / 遺伝子: Pycard, Asc / プラスミド: pET28a / 発現宿主: Escherichia coli BL21(DE3) (大腸菌) / 参照: UniProt: Q9EPB4 |
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-実験情報
-実験
実験 |
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EM実験 | 試料の集合状態: FILAMENT / 3次元再構成法: らせん対称体再構成法 | ||||||||||||||||||||||||||||||||||||||||||||||||||||
NMR実験 |
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-試料調製
構成要素 |
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緩衝液 | 名称: 25 mM Tris, 300 mM sodium chloride / pH: 8 / 詳細: 25 mM Tris, 300 mM sodium chloride | ||||||||||||
試料 | 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES | ||||||||||||
急速凍結 | 装置: FEI VITROBOT MARK IV / 凍結剤: ETHANE 詳細: Grids were blotted for one second before plunging into liquid ethane (FEI VITROBOT MARK IV). | ||||||||||||
詳細 | 内容: 15 mg [U-100% 13C; U-100% 15N] ASC filament, 90% H2O/10% D2O 溶媒系: 90% H2O/10% D2O | ||||||||||||
試料 | 濃度: 15 mg/mL / 構成要素: ASC filament-1 / Isotopic labeling: [U-100% 13C; U-100% 15N] | ||||||||||||
試料状態 | 圧: ambient / 温度: 288 K |
-データ収集
実験機器 | モデル: Titan Krios / 画像提供: FEI Company |
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顕微鏡 | モデル: FEI TITAN KRIOS / 日付: 2014年10月10日 |
電子銃 | 電子線源: TUNGSTEN HAIRPIN / 加速電圧: 300 kV / 照射モード: SPOT SCAN |
電子レンズ | モード: DIFFRACTION回折 / 倍率(公称値): 22500 X |
試料ホルダ | 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER 傾斜角・最大: 12 ° / 傾斜角・最小: -12 ° |
撮影 | 電子線照射量: 20 e/Å2 フィルム・検出器のモデル: DIRECT ELECTRON DE-10 (5k x 4k) |
画像スキャン | デジタル画像の数: 21138 |
NMRスペクトロメーター | タイプ: Bruker Avance / 製造業者: Bruker / モデル: AVANCE / 磁場強度: 850 MHz |
-解析
EMソフトウェア |
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CTF補正 | 詳細: CTFFIND3 | ||||||||||||||||
らせん対称 | 回転角度/サブユニット: 53 ° / 軸方向距離/サブユニット: 14.2 Å / らせん対称軸の対称性: C3 | ||||||||||||||||
3次元再構成 | 手法: layer line analysis / 解像度: 4 Å / 解像度の算出法: FSC 0.5 CUT-OFF / ピクセルサイズ(公称値): 0.67 Å / ピクセルサイズ(実測値): 0.67 Å / 対称性のタイプ: HELICAL | ||||||||||||||||
精密化ステップ | サイクル: LAST
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NMR software |
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精密化 | 手法: simulated annealing / ソフトェア番号: 4 | ||||||||||||||||
代表構造 | 選択基準: lowest energy | ||||||||||||||||
NMRアンサンブル | コンフォーマー選択の基準: structures with the lowest energy 計算したコンフォーマーの数: 100 / 登録したコンフォーマーの数: 10 |