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基本情報
登録情報 | ![]() | ||||||||||||
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タイトル | Arp2/3 branch junction complex, BeFx state | ||||||||||||
![]() | Final cryosparc-refined map, sharpened | ||||||||||||
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機能・相同性 | ![]() Regulation of actin dynamics for phagocytic cup formation / RHO GTPases Activate WASPs and WAVEs / ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() 類似検索 - 分子機能 | ||||||||||||
生物種 | ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() | ||||||||||||
手法 | ![]() ![]() | ||||||||||||
![]() | Chavali SS / Chou SZ / Sindelar CV | ||||||||||||
資金援助 | ![]()
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![]() | ![]() タイトル: Cryo-EM structures reveal how phosphate release from Arp3 weakens actin filament branches formed by Arp2/3 complex. 著者: Sai Shashank Chavali / Steven Z Chou / Wenxiang Cao / Thomas D Pollard / Enrique M De La Cruz / Charles V Sindelar / ![]() 要旨: Arp2/3 complex nucleates branched actin filaments for cell and organelle movements. Here we report a 2.7 Å resolution cryo-EM structure of the mature branch junction formed by S. pombe Arp2/3 ...Arp2/3 complex nucleates branched actin filaments for cell and organelle movements. Here we report a 2.7 Å resolution cryo-EM structure of the mature branch junction formed by S. pombe Arp2/3 complex that provides details about interactions with both mother and daughter filaments. We determine a second structure at 3.2 Å resolution with the phosphate analog BeF bound with ADP to Arp3 and ATP bound to Arp2. In this ADP-BeF transition state the outer domain of Arp3 is rotated 2° toward the mother filament compared with the ADP state and makes slightly broader contacts with actin in both the mother and daughter filaments. Thus, dissociation of P from the ADP-P transition state reduces the interactions of Arp2/3 complex with the actin filaments and may contribute to the lower mechanical stability of mature branch junctions with ADP bound to the Arps. Our structures also reveal that the mother filament in contact with Arp2/3 complex is slightly bent and twisted, consistent with the preference of Arp2/3 complex binding curved actin filaments. The small degree of twisting constrains models of actin filament mechanics. | ||||||||||||
履歴 |
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構造の表示
添付画像 |
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-EMDBアーカイブ
マップデータ | ![]() | 59.4 MB | ![]() | |
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ヘッダ (付随情報) | ![]() ![]() | 33 KB 33 KB | 表示 表示 | ![]() |
FSC (解像度算出) | ![]() | 9.1 KB | 表示 | ![]() |
画像 | ![]() | 144.1 KB | ||
Filedesc metadata | ![]() | 8.9 KB | ||
その他 | ![]() ![]() ![]() | 32.5 MB 59.4 MB 59.4 MB | ||
アーカイブディレクトリ | ![]() ![]() | HTTPS FTP |
-関連構造データ
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リンク
EMDBのページ | ![]() ![]() |
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「今月の分子」の関連する項目 |
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マップ
ファイル | ![]() | ||||||||||||||||||||
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注釈 | Final cryosparc-refined map, sharpened | ||||||||||||||||||||
ボクセルのサイズ | X=Y=Z: 1.346 Å | ||||||||||||||||||||
密度 |
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対称性 | 空間群: 1 | ||||||||||||||||||||
詳細 | EMDB XML:
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-添付データ
-追加マップ: Final cryosparc-refined map, no sharpening
ファイル | emd_42788_additional_1.map | ||||||||||||
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注釈 | Final cryosparc-refined map, no sharpening | ||||||||||||
投影像・断面図 |
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密度ヒストグラム |
-ハーフマップ: cryosparc half-map A
ファイル | emd_42788_half_map_1.map | ||||||||||||
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注釈 | cryosparc half-map A | ||||||||||||
投影像・断面図 |
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密度ヒストグラム |
-ハーフマップ: cryosparc half-map B
ファイル | emd_42788_half_map_2.map | ||||||||||||
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注釈 | cryosparc half-map B | ||||||||||||
投影像・断面図 |
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密度ヒストグラム |
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試料の構成要素
+全体 : Arp2/3 branch complex (ADP-BeFx state)
+超分子 #1: Arp2/3 branch complex (ADP-BeFx state)
+超分子 #2: Arp2/3 complex
+超分子 #3: Actin
+分子 #1: Actin-related protein 3
+分子 #2: Actin-related protein 2
+分子 #3: Actin-related protein 2/3 complex subunit 1
+分子 #4: Actin-related protein 2/3 complex subunit 2
+分子 #5: Actin-related protein 2/3 complex subunit 3
+分子 #6: Actin-related protein 2/3 complex subunit 4
+分子 #7: Actin-related protein 2/3 complex subunit 5
+分子 #8: Actin, alpha skeletal muscle
+分子 #9: ADENOSINE-5'-DIPHOSPHATE
+分子 #10: MAGNESIUM ION
+分子 #11: BERYLLIUM TRIFLUORIDE ION
+分子 #12: ADENOSINE-5'-TRIPHOSPHATE
+分子 #13: water
-実験情報
-構造解析
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試料の集合状態 | particle |
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試料調製
緩衝液 | pH: 7.5 |
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グリッド | モデル: Quantifoil R1.2/1.3 / 材質: GOLD / メッシュ: 300 / 支持フィルム - 材質: GOLD / 支持フィルム - トポロジー: HOLEY / 詳細: Grids were not glow discharged |
凍結 | 凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 277 K / 装置: FEI VITROBOT MARK IV 詳細: The samples were incubated on the grid for 50 s and the extra solution was blotted using two Vitrobot filter papers (0.55/20 mm, Grade 595, Ted Pella) for 4 s at 0 blot force. The grids were ...詳細: The samples were incubated on the grid for 50 s and the extra solution was blotted using two Vitrobot filter papers (0.55/20 mm, Grade 595, Ted Pella) for 4 s at 0 blot force. The grids were plunged into liquid ethane at ~180 degrees C with a wait time of 0.5 s.. |
詳細 | Actin monomers with bound Ca2+ were converted to Mg2+-actin by equilibrating with 50 micromolar MgCl2 and 0.2 mM EGTA (pH 7.5) for 10 min on ice. Actin was polymerized in the presence of capping protein (CP) by sequentially mixing 8.75 micromolar Mg-ATP-actin monomers with 0.75 micromolar CP and equilibrated at room temperature for 1 h. In parallel, Arp2/3 complex (0.4 micromolar) in QB buffer was activated by mixing 0.85 micromolar GCN4-VCA and 50 micromolar ATP and incubated at 4 degrees for 1 h. The capped actin filaments sample was then gently mixed with an equal volume of activated Arp2/3 complex sample using cut pipette tips and equilibrated at 4 degrees for 5 min. Daughter filaments were formed and elongated in the presence of CP by adding 0.25 micromolar Mg-actin monomers, 50 micromolar ATP, and 40 nM CP and incubated for 5 min at room temperature. This step was subsequently repeated 4 more times, then aged for ~90 min before preparing grids for cryo-EM data collection. A final concentration of 2 mM BeFx (prepared using 2 mM BeSO4 and 10 mM NaF) was added during the 4th step of daughter filament formation (as mentioned above). |
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電子顕微鏡法
顕微鏡 | FEI TITAN KRIOS |
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電子線 | 加速電圧: 300 kV / 電子線源: ![]() |
電子光学系 | 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD![]() |
特殊光学系 | エネルギーフィルター - 名称: GIF Quantum LS / エネルギーフィルター - スリット幅: 20 eV 詳細: Electron micrographs for image reconstructions were collected using Titan Krios equipped with X-cold field emission gun at 300 kV, Gatan image filter with slit width of 20 eV and a nanoprobe. |
試料ステージ | 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER ホルダー冷却材: NITROGEN |
詳細 | The vitrified grids were screened for sample homogeneity and ice thickness in a Glacios 200 kV transmission electron microscope equipped with Gatan K2 summit camera. Electron micrographs for image reconstructions were collected using Titan Krios equipped with X-cold field emission gun at 300 kV, Gatan image filter with slit width of 20 eV and a nanoprobe. |
撮影 | フィルム・検出器のモデル: GATAN K3 (6k x 4k) / 実像数: 8519 / 平均露光時間: 3.3 sec. / 平均電子線量: 51.4 e/Å2 詳細: Each movie contains 41 frames with a frame time of 0.08 s. A dose rate of 28.4 counts/pixel/s and a physical pixel size of 1.346 Angstroms was used. |
実験機器 | ![]() モデル: Titan Krios / 画像提供: FEI Company |