National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R01GM127440, R01GM092917, S10OD012272
米国
引用
ジャーナル: Nat Struct Mol Biol / 年: 2020 タイトル: Cryo-EM reveals the transition of Arp2/3 complex from inactive to nucleation-competent state. 著者: Mohammed Shaaban / Saikat Chowdhury / Brad J Nolen / 要旨: Arp2/3 complex, a crucial actin filament nucleator, undergoes structural rearrangements during activation by nucleation-promoting factors (NPFs). However, the conformational pathway leading to the ...Arp2/3 complex, a crucial actin filament nucleator, undergoes structural rearrangements during activation by nucleation-promoting factors (NPFs). However, the conformational pathway leading to the nucleation-competent state is unclear due to lack of high-resolution structures of the activated state. Here we report a ~3.9 Å resolution cryo-EM structure of activated Schizosaccharomyces pombe Arp2/3 complex bound to the S. pombe NPF Dip1 and attached to the end of the nucleated actin filament. The structure reveals global and local conformational changes that allow the two actin-related proteins in Arp2/3 complex to mimic a filamentous actin dimer and template nucleation. Activation occurs through a clamp-twisting mechanism, in which Dip1 forces two core subunits in Arp2/3 complex to pivot around one another, shifting half of the complex into a new activated position. By showing how Dip1 stimulates activation, the structure reveals how NPFs can activate Arp2/3 complex in diverse cellular processes.
凍結剤: ETHANE / チャンバー内湿度: 98 % / チャンバー内温度: 277.15 K / 装置: HOMEMADE PLUNGER 詳細: 4 uL of sample was applied to freshly glow-discharged grid. Excess sample was manually blotted off with a dry Whatman No.1 filter paper for 5-7 seconds. Immediately after the blotting step ...詳細: 4 uL of sample was applied to freshly glow-discharged grid. Excess sample was manually blotted off with a dry Whatman No.1 filter paper for 5-7 seconds. Immediately after the blotting step the sample containing grid was rapidly vitrified by plunge freezing into liquid ethane..
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電子顕微鏡法
顕微鏡
FEI TALOS ARCTICA
詳細
Data were collected by combining untilted and tilted (20, 30 and 40 degrees) images. Stage shifting to the targeted exposure position was used for navigation during data acquisition.
撮影
フィルム・検出器のモデル: FEI FALCON III (4k x 4k) 検出モード: COUNTING / デジタル化 - サイズ - 横: 4000 pixel / デジタル化 - サイズ - 縦: 4000 pixel / 撮影したグリッド数: 3 / 実像数: 5309 / 平均露光時間: 40.0 sec. / 平均電子線量: 44.34 e/Å2 詳細: Each micrograph was collected as dose-fractionated movies consisting of 62 fractions per movie.