+データを開く
-基本情報
登録情報 | データベース: EMDB / ID: EMD-10133 | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
タイトル | cryo-EM structure of mTORC1 bound to active RagA/C GTPases | ||||||||||||
マップデータ | Cryo-EM structure of mTORC1 bound to active RagA/C GTPases. The Final map for the coordinates. | ||||||||||||
試料 |
| ||||||||||||
キーワード | small GTPases / mTORC1 activator / roadblock domain / GTPase domain / SIGNALING PROTEIN | ||||||||||||
機能・相同性 | 機能・相同性情報 Gtr1-Gtr2 GTPase complex / FNIP-folliculin RagC/D GAP / RNA polymerase III type 2 promoter sequence-specific DNA binding / RNA polymerase III type 1 promoter sequence-specific DNA binding / positive regulation of cytoplasmic translational initiation / regulation of locomotor rhythm / T-helper 1 cell lineage commitment / positive regulation of pentose-phosphate shunt / positive regulation of wound healing, spreading of epidermal cells / TORC2 complex ...Gtr1-Gtr2 GTPase complex / FNIP-folliculin RagC/D GAP / RNA polymerase III type 2 promoter sequence-specific DNA binding / RNA polymerase III type 1 promoter sequence-specific DNA binding / positive regulation of cytoplasmic translational initiation / regulation of locomotor rhythm / T-helper 1 cell lineage commitment / positive regulation of pentose-phosphate shunt / positive regulation of wound healing, spreading of epidermal cells / TORC2 complex / cellular response to leucine starvation / TFIIIC-class transcription factor complex binding / regulation of TORC1 signaling / regulation of membrane permeability / negative regulation of lysosome organization / heart valve morphogenesis / nucleus localization / protein localization to lysosome / RNA polymerase III type 3 promoter sequence-specific DNA binding / TORC1 complex / positive regulation of transcription of nucleolar large rRNA by RNA polymerase I / regulation of TOR signaling / calcineurin-NFAT signaling cascade / voluntary musculoskeletal movement / TORC1 signaling / regulation of osteoclast differentiation / positive regulation of odontoblast differentiation / positive regulation of keratinocyte migration / cellular response to L-leucine / Amino acids regulate mTORC1 / MTOR signalling / cellular response to nutrient / regulation of autophagosome assembly / cellular response to methionine / Energy dependent regulation of mTOR by LKB1-AMPK / energy reserve metabolic process / negative regulation of cell size / protein localization to membrane / ruffle organization / positive regulation of osteoclast differentiation / cellular response to osmotic stress / protein serine/threonine kinase inhibitor activity / enzyme-substrate adaptor activity / negative regulation of protein localization to nucleus / anoikis / cardiac muscle cell development / positive regulation of transcription by RNA polymerase III / negative regulation of calcineurin-NFAT signaling cascade / regulation of myelination / positive regulation of actin filament polymerization / small GTPase-mediated signal transduction / regulation of cell size / negative regulation of macroautophagy / positive regulation of oligodendrocyte differentiation / lysosome organization / Macroautophagy / positive regulation of myotube differentiation / protein kinase activator activity / behavioral response to pain / oligodendrocyte differentiation / Constitutive Signaling by AKT1 E17K in Cancer / mTORC1-mediated signalling / germ cell development / : / CD28 dependent PI3K/Akt signaling / social behavior / HSF1-dependent transactivation / neuronal action potential / positive regulation of TOR signaling / response to amino acid / TOR signaling / endomembrane system / 'de novo' pyrimidine nucleobase biosynthetic process / regulation of macroautophagy / positive regulation of translational initiation / positive regulation of G1/S transition of mitotic cell cycle / cellular response to nutrient levels / positive regulation of lamellipodium assembly / phagocytic vesicle / heart morphogenesis / positive regulation of lipid biosynthetic process / positive regulation of epithelial to mesenchymal transition / phosphorylation / cardiac muscle contraction / regulation of cellular response to heat / protein-membrane adaptor activity / positive regulation of stress fiber assembly / tumor necrosis factor-mediated signaling pathway / cytoskeleton organization / 14-3-3 protein binding / positive regulation of endothelial cell proliferation / T cell costimulation / positive regulation of TORC1 signaling / cellular response to amino acid starvation / cellular response to starvation / post-embryonic development / positive regulation of glycolytic process / negative regulation of autophagy / protein serine/threonine kinase activator activity / RNA splicing 類似検索 - 分子機能 | ||||||||||||
生物種 | Homo sapiens (ヒト) | ||||||||||||
手法 | 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 6.2 Å | ||||||||||||
データ登録者 | Anandapadamanaban M / Berndt A | ||||||||||||
資金援助 | 英国, 3件
| ||||||||||||
引用 | ジャーナル: Science / 年: 2019 タイトル: Architecture of human Rag GTPase heterodimers and their complex with mTORC1. 著者: Madhanagopal Anandapadamanaban / Glenn R Masson / Olga Perisic / Alex Berndt / Jonathan Kaufman / Chris M Johnson / Balaji Santhanam / Kacper B Rogala / David M Sabatini / Roger L Williams / 要旨: The Rag guanosine triphosphatases (GTPases) recruit the master kinase mTORC1 to lysosomes to regulate cell growth and proliferation in response to amino acid availability. The nucleotide state of Rag ...The Rag guanosine triphosphatases (GTPases) recruit the master kinase mTORC1 to lysosomes to regulate cell growth and proliferation in response to amino acid availability. The nucleotide state of Rag heterodimers is critical for their association with mTORC1. Our cryo-electron microscopy structure of RagA/RagC in complex with mTORC1 shows the details of RagA/RagC binding to the RAPTOR subunit of mTORC1 and explains why only the RagA/RagC nucleotide state binds mTORC1. Previous kinetic studies suggested that GTP binding to one Rag locks the heterodimer to prevent GTP binding to the other. Our crystal structures and dynamics of RagA/RagC show the mechanism for this locking and explain how oncogenic hotspot mutations disrupt this process. In contrast to allosteric activation by RHEB, Rag heterodimer binding does not change mTORC1 conformation and activates mTORC1 by targeting it to lysosomes. | ||||||||||||
履歴 |
|
-構造の表示
ムービー |
ムービービューア |
---|---|
構造ビューア | EMマップ: SurfViewMolmilJmol/JSmol |
添付画像 |
-ダウンロードとリンク
-EMDBアーカイブ
マップデータ | emd_10133.map.gz | 114.6 MB | EMDBマップデータ形式 | |
---|---|---|---|---|
ヘッダ (付随情報) | emd-10133-v30.xml emd-10133.xml | 33.9 KB 33.9 KB | 表示 表示 | EMDBヘッダ |
FSC (解像度算出) | emd_10133_fsc.xml | 11.4 KB | 表示 | FSCデータファイル |
画像 | emd_10133.png | 263.6 KB | ||
マスクデータ | emd_10133_msk_1.map | 125 MB | マスクマップ | |
Filedesc metadata | emd-10133.cif.gz | 10.8 KB | ||
その他 | emd_10133_additional.map.gz emd_10133_half_map_1.map.gz emd_10133_half_map_2.map.gz | 115.6 MB 98 MB 98.2 MB | ||
アーカイブディレクトリ | http://ftp.pdbj.org/pub/emdb/structures/EMD-10133 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-10133 | HTTPS FTP |
-検証レポート
文書・要旨 | emd_10133_validation.pdf.gz | 947.9 KB | 表示 | EMDB検証レポート |
---|---|---|---|---|
文書・詳細版 | emd_10133_full_validation.pdf.gz | 947.5 KB | 表示 | |
XML形式データ | emd_10133_validation.xml.gz | 18.7 KB | 表示 | |
CIF形式データ | emd_10133_validation.cif.gz | 24.7 KB | 表示 | |
アーカイブディレクトリ | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-10133 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-10133 | HTTPS FTP |
-関連構造データ
-リンク
EMDBのページ | EMDB (EBI/PDBe) / EMDataResource |
---|---|
「今月の分子」の関連する項目 |
-マップ
ファイル | ダウンロード / ファイル: emd_10133.map.gz / 形式: CCP4 / 大きさ: 125 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
注釈 | Cryo-EM structure of mTORC1 bound to active RagA/C GTPases. The Final map for the coordinates. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
投影像・断面図 | 画像のコントロール
画像は Spider により作成 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ボクセルのサイズ | X=Y=Z: 1.43 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
密度 |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
対称性 | 空間群: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
詳細 | EMDB XML:
CCP4マップ ヘッダ情報:
|
-添付データ
-マスク #1
ファイル | emd_10133_msk_1.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
投影像・断面図 |
| ||||||||||||
密度ヒストグラム |
-追加マップ: The experimental EM map for mTORC1-RagA/C monomer after...
ファイル | emd_10133_additional.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
注釈 | The experimental EM map for mTORC1-RagA/C monomer after focussed classification with signal subtraction. | ||||||||||||
投影像・断面図 |
| ||||||||||||
密度ヒストグラム |
-ハーフマップ: Half1 map from the Refine 3D reconstruction (RELION-3.0.6)
ファイル | emd_10133_half_map_1.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
注釈 | Half1 map from the Refine 3D reconstruction (RELION-3.0.6) | ||||||||||||
投影像・断面図 |
| ||||||||||||
密度ヒストグラム |
-ハーフマップ: Half2 map from the Refine 3D reconstruction (RELION-3.0.6)
ファイル | emd_10133_half_map_2.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
注釈 | Half2 map from the Refine 3D reconstruction (RELION-3.0.6) | ||||||||||||
投影像・断面図 |
| ||||||||||||
密度ヒストグラム |
-試料の構成要素
+全体 : cryo-EM structure of mTORC1 bound to active RagA/C complex
+超分子 #1: cryo-EM structure of mTORC1 bound to active RagA/C complex
+超分子 #2: mTORC1
+超分子 #3: RagA/C
+分子 #1: mTOR,Serine/threonine-protein kinase mTOR,mTOR,Serine/threonine-p...
+分子 #2: Target of rapamycin complex subunit LST8
+分子 #3: Ras-related GTP-binding protein A
+分子 #4: Ras-related GTP-binding protein C
+分子 #5: Regulatory-associated protein of mTOR
+分子 #6: GUANOSINE-5'-TRIPHOSPHATE
+分子 #7: GUANOSINE-5'-DIPHOSPHATE
-実験情報
-構造解析
手法 | クライオ電子顕微鏡法 |
---|---|
解析 | 単粒子再構成法 |
試料の集合状態 | particle |
-試料調製
濃度 | 0.05 mg/mL |
---|---|
緩衝液 | pH: 7 詳細: 100mM Tris-HCl pH7.0, 260mM NaCl, 5mM MgCl2, 1mM TCEP |
グリッド | モデル: Quantifoil R1.2/1.3 / 材質: GOLD / メッシュ: 300 |
凍結 | 凍結剤: ETHANE / チャンバー内湿度: 95 % / 装置: FEI VITROBOT MARK III |
詳細 | mTORC1 (mTOR complex 1) is a dimer consists of three proteins: mTOR, mLST8 and RAPTOR. The small GTPases, RagA/C in its active form bind to mTORC1 for activation. We solved the cryo-EM structure of mTORC1 bound to RagA/C. |
-電子顕微鏡法
顕微鏡 | FEI TITAN KRIOS |
---|---|
撮影 | フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) 検出モード: COUNTING / デジタル化 - 画像ごとのフレーム数: 1-22 / 平均露光時間: 1.8 sec. / 平均電子線量: 40.0 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: FLOOD BEAM / 撮影モード: BRIGHT FIELD / Cs: 2.7 mm |
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 |
+画像解析
-原子モデル構築 1
初期モデル |
| ||||||
---|---|---|---|---|---|---|---|
詳細 | Cryo-EM model of mTORC1-RagA/C was refined using REFMAC5 program in CCPEM package, with a composite map of the 3D reconstruction of mTORC1-RagA/C pseudo-monomer (as mentioned in Reconstruction section) of one protomer together with the generated map for the other second protomer of mTORC1-RagA/C. This second protomer of mTORC1-RagA/C map was generated by simply aligning the first 3D reconstructed pseudomonomer map onto the mTORC1 dimer consensus C2 map and then obtained the rotation-translation matrix with CHIMERA and then used Maputils program in CCP4i. From the resulting mTORC1-RagA/C dimer map, the model of mTORC1-RagA/C was built by using previously published structure of apo-mTORC1 (PDB ID 6BCX) and our crystal structure of RagA/C was fitted (PDB ID 6S6A, unreleased). The entire mTORC1-RagA/C final model was refined using REFMAC5 program using the restraints from the crystal structure of RagA/C and previously published mTORC1 structure. Side chains were removed before refinement, since these were not evident in the cryo-EM densities. Separate model refinements were performed against single half-maps, and the resulting models were compared with the other half-maps to confirm the absence of overfitting. | ||||||
精密化 | 空間: REAL / プロトコル: RIGID BODY FIT / 温度因子: 315 | ||||||
得られたモデル | PDB-6sb2: |