Ministry of Education, Culture, Sports, Science and Technology (Japan)
jp15076210
日本
Ministry of Education, Culture, Sports, Science and Technology (Japan)
jp20050030
日本
Ministry of Education, Culture, Sports, Science and Technology (Japan)
jp22018027
日本
Ministry of Education, Culture, Sports, Science and Technology (Japan)
jp23120525
日本
Ministry of Education, Culture, Sports, Science and Technology (Japan)
jp25120725
日本
Ministry of Education, Culture, Sports, Science and Technology (Japan)
jp15H01647
日本
Ministry of Education, Culture, Sports, Science and Technology (Japan)
jp17H05891
日本
引用
ジャーナル: To Be Published タイトル: Mechanism for drastic reduction in catalytic activity of Trp89Phe-mutated glutamate dehydrogenase revealed by crystal structure and cryoEM-sampling of metastable conformation in action 著者: Wakabayashi T / Oide M / Matsui Y / Nakasako M
詳細: 0.5 mM NADP, 100 mM Glutamate in 5 mM Tris-HCl at pH7.5.
グリッド
モデル: Quantifoil R1.2/1.3 / 材質: COPPER / メッシュ: 200 / 支持フィルム - 材質: CARBON / 支持フィルム - トポロジー: HOLEY / 前処理 - タイプ: GLOW DISCHARGE / 前処理 - 時間: 45 sec. / 前処理 - 雰囲気: AIR / 前処理 - 気圧: 0.02 kPa 詳細: Both sides of the grid were glow-discharged for 45 s at 20 mA and 20 Pa.
凍結
凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 281 K / 装置: FEI VITROBOT MARK IV 詳細: The sample solution was flash-frozen 2-h after mixing the protein solution and buffer solution..
詳細
8.96 mg/mL GDH W89F
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電子顕微鏡法
顕微鏡
JEOL CRYO ARM 300
撮影
フィルム・検出器のモデル: GATAN K3 (6k x 4k) / 撮影したグリッド数: 1 / 実像数: 7651 / 平均露光時間: 2.0 sec. / 平均電子線量: 50.0 e/Å2
電子線
加速電圧: 300 kV / 電子線源: FIELD EMISSION GUN
電子光学系
照射モード: OTHER / 撮影モード: BRIGHT FIELD / 最大 デフォーカス(公称値): 5.0 µm 最小 デフォーカス(公称値): 0.35000000000000003 µm
試料ステージ
ホルダー冷却材: NITROGEN
+
画像解析
粒子像選択
選択した数: 3534400
初期モデル
モデルのタイプ: INSILICO MODEL 詳細: The startup model was generated by de novo 3D model generation in Relion.
タイプ: MAXIMUM LIKELIHOOD / ソフトウェア - 名称: RELION (ver. 4.0)
最終 角度割当
タイプ: MAXIMUM LIKELIHOOD / ソフトウェア - 名称: RELION (ver. 4.0)
最終 3次元分類
クラス数: 32 / 平均メンバー数/クラス: 115000 / ソフトウェア - 名称: RELION (ver. 4.0) 詳細: The final 3D classification carried out by two-step process. In the first, all particles were separated into eight classes. Subsequently, each of these classes was further classified into ...詳細: The final 3D classification carried out by two-step process. In the first, all particles were separated into eight classes. Subsequently, each of these classes was further classified into four subclasses. This entire process was conducted following a symmetry expansion operation on all particles, in accordance with the D3 symmetry of GDH.