ジャーナル: Nat Commun / 年: 2021 タイトル: Structure of a microtubule-bound axonemal dynein. 著者: Travis Walton / Hao Wu / Alan Brown / 要旨: Axonemal dyneins are tethered to doublet microtubules inside cilia to drive ciliary beating, a process critical for cellular motility and extracellular fluid flow. Axonemal dyneins are evolutionarily ...Axonemal dyneins are tethered to doublet microtubules inside cilia to drive ciliary beating, a process critical for cellular motility and extracellular fluid flow. Axonemal dyneins are evolutionarily and biochemically distinct from cytoplasmic dyneins that transport cargo, and the mechanisms regulating their localization and function are poorly understood. Here, we report a single-particle cryo-EM reconstruction of a three-headed axonemal dynein natively bound to doublet microtubules isolated from cilia. The slanted conformation of the axonemal dynein causes interaction of its motor domains with the neighboring dynein complex. Our structure shows how a heterotrimeric docking complex specifically localizes the linear array of axonemal dyneins to the doublet microtubule by directly interacting with the heavy chains. Our structural analysis establishes the arrangement of conserved heavy, intermediate and light chain subunits, and provides a framework to understand the roles of individual subunits and the interactions between dyneins during ciliary waveform generation.
ChemComp-GDP: GUANOSINE-5'-DIPHOSPHATE / GDP / GDP, エネルギー貯蔵分子*YM
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実験情報
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構造解析
手法
クライオ電子顕微鏡法
解析
らせん対称体再構成法
試料の集合状態
filament
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試料調製
濃度
20 mg/mL
緩衝液
pH: 7.4 構成要素:
濃度
名称
式
30.0 mM
HEPES
25.0 mM
KCl
5.0 mM
MgSO4
0.5 mM
EGTA
10.0 mM
ATP
0.75 mM
CaCl2
詳細: Buffer also contained 1x Protease Arrest (G-Biosciences)
グリッド
前処理 - タイプ: GLOW DISCHARGE / 前処理 - 時間: 30 sec. / 詳細: 15 mA
凍結
凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 298 K / 装置: FEI VITROBOT MARK IV 詳細: 2.5 ul of splayed axoneme solution was then dispensed onto glow-discharged C-Flat 1.2/1.3-4Cu grids inside a Vitrobot Mark IV under 100% humidity. After a 10 s delay time, cryo-EM samples ...詳細: 2.5 ul of splayed axoneme solution was then dispensed onto glow-discharged C-Flat 1.2/1.3-4Cu grids inside a Vitrobot Mark IV under 100% humidity. After a 10 s delay time, cryo-EM samples were prepared by first blotting for 10 s with blot force set to 16 and immediately plunged into liquid ethane..
詳細
Splayed axonemes isolated from Chlamydomonas reinhardtii flagella.