National Institutes of Health/National Cancer Institute (NIH/NCI)
RO1 CA69202
米国
引用
ジャーナル: iScience / 年: 2021 タイトル: Cryo-electron microscopy structures of VCP/p97 reveal a new mechanism of oligomerization regulation. 著者: Guimei Yu / Yunpeng Bai / Kunpeng Li / Ovini Amarasinghe / Wen Jiang / Zhong-Yin Zhang / 要旨: VCP/p97 is an evolutionarily conserved AAA+ ATPase important for cellular homeostasis. Previous studies suggest that VCP predominantly exists as a homohexamer. Here, we performed structural and ...VCP/p97 is an evolutionarily conserved AAA+ ATPase important for cellular homeostasis. Previous studies suggest that VCP predominantly exists as a homohexamer. Here, we performed structural and biochemical characterization of VCP dodecamer, an understudied state of VCP. The structure revealed an apo nucleotide status that has rarely been captured, a tail-to-tail assembly of two hexamers, and the up-elevated N-terminal domains akin to that seen in the ATP-bound hexamer. Further analyses elucidated a nucleotide status-dependent dodecamerization mechanism, where nucleotide dissociation from the D2 AAA domains induces and promotes VCP dodecamerization. In contrast, nucleotide-free D1 AAA domains are associated with the up-rotation of N-terminal domains, which may prime D1 for ATP binding. These results therefore reveal new nucleotide status-dictated intra- and interhexamer conformational changes and suggest that modulation of D2 domain nucleotide occupancy may serve as a mechanism in controlling VCP oligomeric states.
モデル: PELCO Ultrathin Carbon with Lacey Carbon / 材質: COPPER / メッシュ: 400 / 支持フィルム - #0 - Film type ID: 1 / 支持フィルム - #0 - 材質: CARBON / 支持フィルム - #0 - トポロジー: LACEY / 支持フィルム - #1 - Film type ID: 2 / 支持フィルム - #1 - 材質: GRAPHENE OXIDE / 支持フィルム - #1 - トポロジー: CONTINUOUS / 前処理 - タイプ: GLOW DISCHARGE / 前処理 - 時間: 40 sec. / 前処理 - 雰囲気: AIR / 詳細: 15mA for 40s using Emitech.
凍結
凍結剤: ETHANE / チャンバー内湿度: 70 % / チャンバー内温度: 293 K / 装置: GATAN CRYOPLUNGE 3 詳細: 3.5ul sample was applied to a lacy carbon grid coated with graphene oxide. 7 seconds blot with filter paper was performed using Gatan Cp3..
詳細
VCP/p97 was expressed and purified from H1299 cells using anti-Flag resin and 3XFlag peptide.
フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) 検出モード: SUPER-RESOLUTION / 撮影したグリッド数: 1 / 実像数: 600 / 平均露光時間: 10.0 sec. / 平均電子線量: 46.0 e/Å2 詳細: A dataset of ~600 movies was collected using FEI Titan Krios TEM operating at 300 KV and recorded with the Gatan K2 Summit direct electron detector at super-resolution mode with 22500 nominal ...詳細: A dataset of ~600 movies was collected using FEI Titan Krios TEM operating at 300 KV and recorded with the Gatan K2 Summit direct electron detector at super-resolution mode with 22500 nominal magnification. A frame rate of 5 frames per second, a dose rate of 8 eps and a total exposure of 10 seconds were used.
実験機器
モデル: Titan Krios / 画像提供: FEI Company
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画像解析
粒子像選択
選択した数: 40000
初期モデル
モデルのタイプ: NONE 詳細: random initial models were generated in cryoSPARC from data.
初期 角度割当
タイプ: RANDOM ASSIGNMENT / ソフトウェア - 名称: cryoSPARC
最終 3次元分類
クラス数: 3 / ソフトウェア - 名称: cryoSPARC 詳細: 27,500 particles were classified to class1, 8,800 particles to class 2 and 3,800 particles to class 3. Further processing and analysis were performed with class 1 with best quality.