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基本情報
登録情報 | データベース: PDB / ID: 4v4l | |||||||||
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タイトル | Structure of the Drosophila apoptosome | |||||||||
![]() | Apaf-1 related killer DARK | |||||||||
![]() | APOPTOSIS / Drosophila apoptosome / programmed cell death | |||||||||
機能・相同性 | ![]() negative regulation of humoral immune response / positive regulation of glial cell apoptotic process / Formation of apoptosome / salivary gland histolysis / positive regulation of compound eye retinal cell programmed cell death / melanization defense response / sarcosine catabolic process / Activation of caspases through apoptosome-mediated cleavage / Regulation of the apoptosome activity / central nervous system formation ...negative regulation of humoral immune response / positive regulation of glial cell apoptotic process / Formation of apoptosome / salivary gland histolysis / positive regulation of compound eye retinal cell programmed cell death / melanization defense response / sarcosine catabolic process / Activation of caspases through apoptosome-mediated cleavage / Regulation of the apoptosome activity / central nervous system formation / chaeta development / sperm individualization / apoptosome / autophagic cell death / Neutrophil degranulation / S-adenosylmethionine cycle / CARD domain binding / programmed cell death / triglyceride homeostasis / dendrite morphogenesis / response to starvation / cysteine-type endopeptidase activator activity involved in apoptotic process / response to gamma radiation / ADP binding / neuron cellular homeostasis / positive regulation of apoptotic process / ATP binding / identical protein binding 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: 6.9 Å | |||||||||
![]() | Yuan, S. / Topf, M. / Akey, C.W. / Ludtke, S.J. | |||||||||
![]() | ![]() タイトル: Structure of the Drosophila apoptosome at 6.9 å resolution. 著者: Shujun Yuan / Xinchao Yu / Maya Topf / Loretta Dorstyn / Sharad Kumar / Steven J Ludtke / Christopher W Akey / ![]() 要旨: The Drosophila Apaf-1 related killer forms an apoptosome in the intrinsic cell death pathway. In this study we show that Dark forms a single ring when initiator procaspases are bound. This Dark-Dronc ...The Drosophila Apaf-1 related killer forms an apoptosome in the intrinsic cell death pathway. In this study we show that Dark forms a single ring when initiator procaspases are bound. This Dark-Dronc complex cleaves DrICE efficiently; hence, a single ring represents the Drosophila apoptosome. We then determined the 3D structure of a double ring at ∼6.9 Å resolution and created a model of the apoptosome. Subunit interactions in the Dark complex are similar to those in Apaf-1 and CED-4 apoptosomes, but there are significant differences. In particular, Dark has "lost" a loop in the nucleotide-binding pocket, which opens a path for possible dATP exchange in the apoptosome. In addition, caspase recruitment domains (CARDs) form a crown on the central hub of the Dark apoptosome. This CARD geometry suggests that conformational changes will be required to form active Dark-Dronc complexes. When taken together, these data provide insights into apoptosome structure, function, and evolution. | |||||||||
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 | ![]() | 2.7 MB | 表示 | ![]() |
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-関連構造データ
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集合体
登録構造単位 | ![]()
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対称性 | 点対称性: (シェーンフリース記号: D8 (2回x8回 2面回転対称)) |
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要素
#1: タンパク質 | 分子量: 123195.531 Da / 分子数: 16 / 由来タイプ: 組換発現 由来: (組換発現) ![]() ![]() 遺伝子: Ark, CG6829, dapaf-1L, Dmel_CG6829, Hac1 / プラスミド: pFastBac 発現宿主: ![]() ![]() 株 (発現宿主): sf21 / 参照: UniProt: Q7KLI1 #2: 化合物 | ChemComp-MG / #3: 化合物 | ChemComp-DTP / 配列の詳細 | AUTHORS STATE THAT THE ACTUAL SEQUENCE FOR THE PROTEIN IS: ...AUTHORS STATE THAT THE ACTUAL SEQUENCE FOR THE PROTEIN IS: MDFETGEHQY | |
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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 |
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分子量 | 値: 2.5 MDa / 実験値: NO | |||||||||||||||
緩衝液 | 名称: HEPES buffer / pH: 7.5 詳細: 20mM HEPES, 10mM KCl, 1.5mM MgCl2, 1mM EDTA, 1mM EGTA, 1mM DTT | |||||||||||||||
試料 | 濃度: 2 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES 詳細: 20mM HEPES, 10mM KCl, 1.5mM MgCl2, 1mM EDTA, 1mM EGTA, 1mM DTT | |||||||||||||||
試料支持 | 詳細: C-flat 2/1 holey grids (400 mesh) covered with a thin carbon film | |||||||||||||||
急速凍結 | 装置: FEI VITROBOT MARK III / 凍結剤: ETHANE / Temp: 77 K / 湿度: 100 % 詳細: blotting at room temperature with sample at room temperature 手法: Blot for 2-2.5s before plunging |
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電子顕微鏡撮影
実験機器 | ![]() モデル: Tecnai F20 / 画像提供: FEI Company |
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顕微鏡 | モデル: FEI TECNAI F20 / 日付: 2009年9月15日 詳細: actual magnification at the ccd 87000, camera pixel size 15um, 1.72 angstrom per pixel, data collected semi-automatically with EMTools (TVIPS) |
電子銃 | 電子線源: ![]() |
電子レンズ | モード: BRIGHT FIELD / 倍率(公称値): 50000 X / 倍率(補正後): 50000 X / 最大 デフォーカス(公称値): 3000 nm / 最小 デフォーカス(公称値): 1500 nm / Cs: 2 mm 非点収差: objective lens astigmatism was corrected at 200,000 times magnification カメラ長: 0 mm |
試料ホルダ | 試料ホルダーモデル: GATAN LIQUID NITROGEN 資料ホルダタイプ: Side entry liquid nitrogen-cooled cryo specimen holder 温度: 93 K / 最高温度: 100 K / 最低温度: 93 K / 傾斜角・最大: 0 ° / 傾斜角・最小: 0 ° |
撮影 | 電子線照射量: 20 e/Å2 フィルム・検出器のモデル: GENERIC TVIPS (4k x 4k) |
放射 | プロトコル: SINGLE WAVELENGTH / 単色(M)・ラウエ(L): M / 散乱光タイプ: x-ray |
放射波長 | 相対比: 1 |
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解析
EMソフトウェア |
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CTF補正 | 詳細: each CCD frame | ||||||||||||||||||||
対称性 | 点対称性: D8 (2回x8回 2面回転対称) | ||||||||||||||||||||
3次元再構成 | 手法: projection matching / 解像度: 6.9 Å / 解像度の算出法: FSC 0.5 CUT-OFF / 粒子像の数: 48271 / ピクセルサイズ(実測値): 1.72 Å 詳細: Projection matching was done with Fourier ring correlation, model-based masking and SSNR weighting over an 80-6 angstrom resolution range. The final refinement steps used an angular step of 2. ...詳細: Projection matching was done with Fourier ring correlation, model-based masking and SSNR weighting over an 80-6 angstrom resolution range. The final refinement steps used an angular step of 2.5 degrees and each of the 48,000 particles was matched to the best two projection classes (1353). In total, 45,000 particles were used in the final reconstruction and the 3D map was amplitude corrected then Gaussian low-pass filtered with a Fourier half-width of 0.12. クラス平均像の数: 1353 / 対称性のタイプ: POINT | ||||||||||||||||||||
原子モデル構築 | プロトコル: FLEXIBLE FIT / 空間: REAL / Target criteria: Cross-correlation coefficient 詳細: METHOD--Flexible fitting, local refinement REFINEMENT PROTOCOL--rigid body first, and then used individual helices DETAILS--Chimera was used to do initial domain fitting with Apaf-1 (1Z6T) ...詳細: METHOD--Flexible fitting, local refinement REFINEMENT PROTOCOL--rigid body first, and then used individual helices DETAILS--Chimera was used to do initial domain fitting with Apaf-1 (1Z6T) and CED-4 (2A5Y)domains. Modeller and Flex-Em were then used to do local refinement of homology models of the various domains. | ||||||||||||||||||||
精密化ステップ | サイクル: LAST
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