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基本情報
登録情報 | データベース: PDB / ID: 8uo4 | |||||||||
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タイトル | CryoEM structure of beta-2-adrenergic receptor in complex with GTP-bound Gs heterotrimer (Class T) | |||||||||
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![]() | SIGNALING PROTEIN / GPCR / Adrenergic / Receptor / G protein | |||||||||
機能・相同性 | ![]() beta2-adrenergic receptor activity / positive regulation of mini excitatory postsynaptic potential / positive regulation of cAMP-dependent protein kinase activity / positive regulation of AMPA receptor activity / norepinephrine binding / Adrenoceptors / heat generation / positive regulation of autophagosome maturation / norepinephrine-epinephrine-mediated vasodilation involved in regulation of systemic arterial blood pressure / activation of transmembrane receptor protein tyrosine kinase activity ...beta2-adrenergic receptor activity / positive regulation of mini excitatory postsynaptic potential / positive regulation of cAMP-dependent protein kinase activity / positive regulation of AMPA receptor activity / norepinephrine binding / Adrenoceptors / heat generation / positive regulation of autophagosome maturation / norepinephrine-epinephrine-mediated vasodilation involved in regulation of systemic arterial blood pressure / activation of transmembrane receptor protein tyrosine kinase activity / negative regulation of smooth muscle contraction / positive regulation of lipophagy / response to psychosocial stress / negative regulation of multicellular organism growth / negative regulation of G protein-coupled receptor signaling pathway / endosome to lysosome transport / adrenergic receptor signaling pathway / diet induced thermogenesis / neuronal dense core vesicle / positive regulation of protein kinase A signaling / PKA activation in glucagon signalling / hair follicle placode formation / adenylate cyclase binding / smooth muscle contraction / developmental growth / D1 dopamine receptor binding / intracellular transport / potassium channel regulator activity / Hedgehog 'off' state / bone resorption / positive regulation of bone mineralization / adenylate cyclase-activating adrenergic receptor signaling pathway / brown fat cell differentiation / activation of adenylate cyclase activity / regulation of sodium ion transport / adenylate cyclase activator activity / response to cold / receptor-mediated endocytosis / trans-Golgi network membrane / clathrin-coated endocytic vesicle membrane / G-protein beta/gamma-subunit complex binding / bone development / Olfactory Signaling Pathway / Activation of the phototransduction cascade / positive regulation of protein serine/threonine kinase activity / adenylate cyclase-modulating G protein-coupled receptor signaling pathway / G beta:gamma signalling through PLC beta / Presynaptic function of Kainate receptors / Thromboxane signalling through TP receptor / adenylate cyclase-activating G protein-coupled receptor signaling pathway / G protein-coupled acetylcholine receptor signaling pathway / G-protein activation / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / Prostacyclin signalling through prostacyclin receptor / Glucagon signaling in metabolic regulation / G beta:gamma signalling through CDC42 / cognition / platelet aggregation / ADP signalling through P2Y purinoceptor 12 / G beta:gamma signalling through BTK / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / Sensory perception of sweet, bitter, and umami (glutamate) taste / photoreceptor disc membrane / Adrenaline,noradrenaline inhibits insulin secretion / Glucagon-type ligand receptors / Vasopressin regulates renal water homeostasis via Aquaporins / G alpha (z) signalling events / cellular response to catecholamine stimulus / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / ADORA2B mediated anti-inflammatory cytokines production / sensory perception of taste / ADP signalling through P2Y purinoceptor 1 / adenylate cyclase-activating dopamine receptor signaling pathway / cellular response to amyloid-beta / G beta:gamma signalling through PI3Kgamma / cellular response to prostaglandin E stimulus / Cooperation of PDCL (PhLP1) and TRiC/CCT in G-protein beta folding / GPER1 signaling / G-protein beta-subunit binding / Inactivation, recovery and regulation of the phototransduction cascade / heterotrimeric G-protein complex / G alpha (12/13) signalling events / extracellular vesicle / sensory perception of smell / signaling receptor complex adaptor activity / Thrombin signalling through proteinase activated receptors (PARs) / Cargo recognition for clathrin-mediated endocytosis / GTPase binding / retina development in camera-type eye / Clathrin-mediated endocytosis / Ca2+ pathway / positive regulation of cold-induced thermogenesis / phospholipase C-activating G protein-coupled receptor signaling pathway / amyloid-beta binding / G alpha (i) signalling events / fibroblast proliferation / G alpha (s) signalling events / G alpha (q) signalling events / Ras protein signal transduction 類似検索 - 分子機能 | |||||||||
生物種 | ![]() | |||||||||
手法 | 電子顕微鏡法 / 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 4 Å | |||||||||
![]() | Papasergi-Scott, M.M. / 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
構造ビューア | 分子: ![]() ![]() |
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ダウンロード
PDBx/mmCIF形式 | ![]() | 168 KB | 表示 | ![]() |
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PDB形式 | ![]() | 114 KB | 表示 | ![]() |
PDBx/mmJSON形式 | ![]() | ツリー表示 | ![]() | |
その他 | ![]() |
-検証レポート
アーカイブディレクトリ | ![]() ![]() | HTTPS FTP |
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-関連構造データ
関連構造データ | ![]() 42427MC ![]() 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 M: このデータのモデリングに利用したマップデータ C: 同じ文献を引用 ( |
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類似構造データ | 類似検索 - 機能・相同性 ![]() |
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リンク
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集合体
登録構造単位 | ![]()
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要素
-Guanine nucleotide-binding protein ... , 3種, 3分子 ABG
#1: タンパク質 | 分子量: 44326.160 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) ![]() ![]() |
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#2: タンパク質 | 分子量: 37573.988 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) ![]() ![]() |
#3: タンパク質 | 分子量: 7861.143 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) ![]() ![]() |
-タンパク質 , 1種, 1分子 R
#4: タンパク質 | 分子量: 51767.242 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) ![]() 発現宿主: ![]() ![]() 参照: UniProt: P07550 |
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-非ポリマー , 3種, 3分子 ![](data/chem/img/GTP.gif)
![](data/chem/img/MG.gif)
![](data/chem/img/G1I.gif)
![](data/chem/img/MG.gif)
![](data/chem/img/G1I.gif)
#5: 化合物 | ChemComp-GTP / |
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#6: 化合物 | ChemComp-MG / |
#7: 化合物 | ChemComp-G1I / ( |
-詳細
研究の焦点であるリガンドがあるか | N |
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-実験情報
-実験
実験 | 手法: 電子顕微鏡法 |
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EM実験 | 試料の集合状態: PARTICLE / 3次元再構成法: 単粒子再構成法 |
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試料調製
構成要素 | 名称: Complex of beta-2 adrenergic receptor and Gs heterotrimer with GTP タイプ: COMPLEX 詳細: Combined datasets of complex plunge frozen at 5 sec, 10 sec, or 17 sec after GTP addition to the sample. Entity ID: #1-#4 / 由来: RECOMBINANT | ||||||||||||
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分子量 | 実験値: NO | ||||||||||||
由来(天然) | 生物種: ![]() | ||||||||||||
由来(組換発現) |
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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. | ||||||||||||
試料 | 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES | ||||||||||||
急速凍結 | 装置: FEI VITROBOT MARK IV / 凍結剤: ETHANE / 湿度: 100 % / 凍結前の試料温度: 277.15 K 詳細: GTP was added just prior to freezing at 5 sec, 10 sec, or 17 sec before plunging. |
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電子顕微鏡撮影
実験機器 | ![]() モデル: Titan Krios / 画像提供: FEI Company |
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顕微鏡 | モデル: FEI TITAN KRIOS |
電子銃 | 電子線源: ![]() |
電子レンズ | モード: BRIGHT FIELD / 最大 デフォーカス(公称値): 2000 nm / 最小 デフォーカス(公称値): 400 nm |
試料ホルダ | 凍結剤: NITROGEN 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER |
撮影 | 電子線照射量: 50 e/Å2 / 検出モード: COUNTING / フィルム・検出器のモデル: GATAN K3 (6k x 4k) 詳細: The cryoEM 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 ...詳細: The cryoEM 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. |
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解析
EMソフトウェア |
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CTF補正 | タイプ: NONE | ||||||||||||||||||||||||||||
粒子像の選択 | 選択した粒子像数: 19918625 | ||||||||||||||||||||||||||||
3次元再構成 | 解像度: 4 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 45805 詳細: Contribution of each dataset to this refined class: 15.00% 5 sec dataset; 49.20% 10 sec dataset; 35.80% 17 sec dataset 対称性のタイプ: POINT | ||||||||||||||||||||||||||||
拘束条件 |
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