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データを開く
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基本情報
登録情報 | ![]() | |||||||||
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タイトル | Locally refined cryoEM structure of G protein heterotrimer from beta-2-adrenergic receptor in complex with GTP-bound Gs heterotrimer (transition intermediate #1 of 20) | |||||||||
![]() | Sharpened map | |||||||||
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![]() | GPCR / Adrenergic / Receptor / G protein / SIGNALING PROTEIN | |||||||||
生物種 | ![]() | |||||||||
手法 | 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 3.1 Å | |||||||||
![]() | Papasergi-Scott MM / Skiniotis G | |||||||||
資金援助 | ![]()
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![]() | ![]() タイトル: Time-resolved cryo-EM of G-protein activation by a GPCR. 著者: Makaía M Papasergi-Scott / Guillermo Pérez-Hernández / Hossein Batebi / Yang Gao / Gözde Eskici / Alpay B Seven / Ouliana Panova / Daniel Hilger / Marina Casiraghi / Feng He / Luis Maul / ...著者: Makaía M Papasergi-Scott / Guillermo Pérez-Hernández / Hossein Batebi / Yang Gao / Gözde Eskici / Alpay B Seven / Ouliana Panova / Daniel Hilger / Marina Casiraghi / Feng He / Luis Maul / Peter Gmeiner / Brian K Kobilka / Peter W Hildebrand / Georgios Skiniotis / ![]() ![]() ![]() 要旨: G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating guanine nucleotide exchange in the Gα subunit. To visualize this mechanism, we developed a time-resolved cryo-EM ...G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating guanine nucleotide exchange in the Gα subunit. To visualize this mechanism, we developed a time-resolved cryo-EM approach that examines the progression of ensembles of pre-steady-state intermediates of a GPCR-G-protein complex. By monitoring the transitions of the stimulatory G protein in complex with the β-adrenergic receptor at short sequential time points after GTP addition, we identified the conformational trajectory underlying G-protein activation and functional dissociation from the receptor. Twenty structures generated from sequential overlapping particle subsets along this trajectory, compared to control structures, provide a high-resolution description of the order of main events driving G-protein activation in response to GTP binding. Structural changes propagate from the nucleotide-binding pocket and extend through the GTPase domain, enacting alterations to Gα switch regions and the α5 helix that weaken the G-protein-receptor interface. Molecular dynamics simulations with late structures in the cryo-EM trajectory support that enhanced ordering of GTP on closure of the α-helical domain against the nucleotide-bound Ras-homology domain correlates with α5 helix destabilization and eventual dissociation of the G protein from the GPCR. These findings also highlight the potential of time-resolved cryo-EM as a tool for mechanistic dissection of GPCR signalling events. | |||||||||
履歴 |
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構造の表示
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ダウンロードとリンク
-EMDBアーカイブ
マップデータ | ![]() | 127.1 MB | ![]() | |
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ヘッダ (付随情報) | ![]() ![]() | 18.4 KB 18.4 KB | 表示 表示 | ![]() |
FSC (解像度算出) | ![]() | 10.8 KB | 表示 | ![]() |
画像 | ![]() | 113.1 KB | ||
マスクデータ | ![]() | 134.6 MB | ![]() | |
Filedesc metadata | ![]() | 5 KB | ||
その他 | ![]() ![]() ![]() | 67.4 MB 125.1 MB 125.1 MB | ||
アーカイブディレクトリ | ![]() ![]() | HTTPS FTP |
-関連構造データ
関連構造データ | ![]() 8gdzC ![]() 8ge1C ![]() 8ge2C ![]() 8ge3C ![]() 8ge4C ![]() 8ge5C ![]() 8ge6C ![]() 8ge7C ![]() 8ge8C ![]() 8ge9C ![]() 8geaC ![]() 8gebC ![]() 8gecC ![]() 8gedC ![]() 8geeC ![]() 8gefC ![]() 8gegC ![]() 8gehC ![]() 8geiC ![]() 8gejC ![]() 8gfvC ![]() 8gfwC ![]() 8gfxC ![]() 8gfyC ![]() 8gfzC ![]() 8gg0C ![]() 8gg1C ![]() 8gg2C ![]() 8gg3C ![]() 8gg4C ![]() 8gg5C ![]() 8gg6C ![]() 8gg7C ![]() 8gg8C ![]() 8gg9C ![]() 8ggaC ![]() 8ggbC ![]() 8ggcC ![]() 8ggeC ![]() 8ggfC ![]() 8ggiC ![]() 8ggjC ![]() 8ggkC ![]() 8gglC ![]() 8ggmC ![]() 8ggnC ![]() 8ggoC ![]() 8ggpC ![]() 8ggqC ![]() 8ggrC ![]() 8ggsC ![]() 8ggtC ![]() 8gguC ![]() 8ggvC ![]() 8ggwC ![]() 8ggxC ![]() 8ggyC ![]() 8ggzC ![]() 8gh0C ![]() 8gh1C ![]() 8unlC ![]() 8unmC ![]() 8unnC ![]() 8unoC ![]() 8unpC ![]() 8unqC ![]() 8unrC ![]() 8unsC ![]() 8untC ![]() 8unuC ![]() 8unvC ![]() 8unwC ![]() 8unxC ![]() 8unyC ![]() 8unzC ![]() 8uo0C ![]() 8uo1C ![]() 8uo2C ![]() 8uo3C ![]() 8uo4C C: 同じ文献を引用 ( |
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リンク
EMDBのページ | ![]() ![]() |
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マップ
ファイル | ![]() | ||||||||||||||||||||
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注釈 | Sharpened map | ||||||||||||||||||||
ボクセルのサイズ | X=Y=Z: 0.8677 Å | ||||||||||||||||||||
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対称性 | 空間群: 1 | ||||||||||||||||||||
詳細 | EMDB XML:
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-添付データ
-マスク #1
ファイル | ![]() | ||||||||||||
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密度ヒストグラム |
-追加マップ: Unsharpened map
ファイル | emd_40156_additional_1.map | ||||||||||||
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注釈 | Unsharpened map | ||||||||||||
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密度ヒストグラム |
-ハーフマップ: Half map
ファイル | emd_40156_half_map_1.map | ||||||||||||
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注釈 | Half map | ||||||||||||
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密度ヒストグラム |
-ハーフマップ: Half map
ファイル | emd_40156_half_map_2.map | ||||||||||||
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注釈 | Half map | ||||||||||||
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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 : Complex of beta-2 adrenergic receptor and Gs heterotrimer with GTP
全体 | 名称: Complex of beta-2 adrenergic receptor and Gs heterotrimer with GTP |
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要素 |
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-超分子 #1: Complex of beta-2 adrenergic receptor and Gs heterotrimer with GTP
超分子 | 名称: Complex of beta-2 adrenergic receptor and Gs heterotrimer with GTP タイプ: complex / ID: 1 / 親要素: 0 詳細: Combined datasets of the protein complex from samples plunge frozen at 5 sec, 10 sec, or 17 sec after GTP addition to the sample. |
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由来(天然) | 生物種: ![]() |
-実験情報
-構造解析
手法 | クライオ電子顕微鏡法 |
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![]() | 単粒子再構成法 |
試料の集合状態 | particle |
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試料調製
緩衝液 | pH: 7.5 詳細: GTP was added just prior to freezing at 5 sec, 10 sec, or 17 sec before plunging. |
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グリッド | モデル: UltrAuFoil R1.2/1.3 / 前処理 - タイプ: GLOW DISCHARGE |
凍結 | 凍結剤: ETHANE / チャンバー内湿度: 100 % / 装置: FEI VITROBOT MARK IV 詳細: GTP was added just prior to freezing at 5 sec, 10 sec, or 17 sec before plunging.. |
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電子顕微鏡法
顕微鏡 | FEI TITAN KRIOS |
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撮影 | フィルム・検出器のモデル: GATAN K3 (6k x 4k) / 平均電子線量: 50.0 e/Å2 詳細: The map is the result of combining multiple datasets: cryo-EM imaging of the beta-2AR-Gs + GTP (5 sec) complex was performed on a Titan Krios electron microscope equipped with a K3 Summit ...詳細: The map is the result of combining multiple datasets: cryo-EM imaging of the beta-2AR-Gs + GTP (5 sec) complex was performed on a Titan Krios electron microscope equipped with a K3 Summit direct electron detector (Gatan). The microscope was operated at 300 kV accelerating voltage, with a nominal magnification of 105,000x in counting mode resulting in a magnified pixel size of 0.8677 Angstrom. A total exposure of 60.48 electrons/ Angstrom^2 over 63 frames with defocus ranging from -1.0 - -2.0 micrometers was used. Cryo-EM imaging of beta-2AR-Gs + GTP (10 sec) complex was performed on four separate grids over three collection sessions. The microscope was operated at 300 kV accelerating voltage, with a magnification at the camera of 58,679x in counting mode resulting in a magnified pixel size of 0.8521 Angstrom. For the first and second grids, movies were obtained at an exposure rate of 21.13 electrons/Angstrum^2/sec with defocus ranging from -0.4 - -2.0 micrometers. The total exposure time was 2.717 sec over 77 frames per movie stack. For an additional collection of the first grid, movies were obtained at an exposure rate of 20.95 electrons/ Angstrum^2/sec with defocus ranging from -0.4 -2.0 micrometers. The total exposure time was 2.717 sec over 77 frames per movie stack. For the third and fourth grids, movies were obtained at an exposure rate of 30.71 electrons/ Angstrum^2/sec with defocus ranging from -0.5 - -1.6 micrometers. The total exposure time was 2.008 sec over 79 frames per movie stack. Cryo-EM imaging of beta-2AR-Gs + GTP (17 sec) was performed on a Titan Krios equipped with a post-column energy filter, with a magnification of 105,000x in counting mode resulting in a magnified pixel size of 0.8677 Angstrom. Movies were obtained at an exposure rate of 32.46 electrons/Angstrum^2/sec with defocus ranging from -0.4 - -0.9 micrometers. The total exposure time was 1.999 sec over 79 frames per movie stack. |
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 / 最大 デフォーカス(公称値): 2.0 µm / 最小 デフォーカス(公称値): 0.4 µm |
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 |
実験機器 | ![]() モデル: Titan Krios / 画像提供: FEI Company |