+データを開く
-基本情報
登録情報 | データベース: PDB / ID: 8vsu | ||||||
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タイトル | Cryo-EM structure of LKB1-STRADalpha-MO25alpha heterocomplex | ||||||
要素 |
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キーワード | TRANSFERASE / serine/threonine kinase / pseudokinase / complex | ||||||
機能・相同性 | 機能・相同性情報 positive regulation of vesicle transport along microtubule / intracellular protein-containing complex / Golgi localization / LRR domain binding / negative regulation of potassium ion transmembrane transporter activity / AMPK inhibits chREBP transcriptional activation activity / negative regulation of potassium ion transmembrane transport / dendrite extension / negative regulation of epithelial cell proliferation involved in prostate gland development / serine/threonine protein kinase complex ...positive regulation of vesicle transport along microtubule / intracellular protein-containing complex / Golgi localization / LRR domain binding / negative regulation of potassium ion transmembrane transporter activity / AMPK inhibits chREBP transcriptional activation activity / negative regulation of potassium ion transmembrane transport / dendrite extension / negative regulation of epithelial cell proliferation involved in prostate gland development / serine/threonine protein kinase complex / cellular hypotonic response / tissue homeostasis / epithelial cell proliferation involved in prostate gland development / response to thyroid hormone / Energy dependent regulation of mTOR by LKB1-AMPK / positive thymic T cell selection / vasculature development / regulation of Wnt signaling pathway / anoikis / response to glucagon / negative regulation of cold-induced thermogenesis / FOXO-mediated transcription of cell death genes / positive regulation of axonogenesis / establishment of cell polarity / cellular response to UV-B / regulation of dendrite morphogenesis / response to ionizing radiation / G1 to G0 transition / response to lipid / activation of protein kinase activity / protein kinase activator activity / regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / positive regulation of transforming growth factor beta receptor signaling pathway / intrinsic apoptotic signaling pathway by p53 class mediator / protein localization to nucleus / positive regulation of autophagy / positive regulation of gluconeogenesis / negative regulation of TORC1 signaling / protein export from nucleus / protein serine/threonine kinase activator activity / protein dephosphorylation / axonogenesis / positive regulation of peptidyl-threonine phosphorylation / regulation of signal transduction by p53 class mediator / response to activity / regulation of cell growth / peptidyl-threonine phosphorylation / negative regulation of canonical Wnt signaling pathway / positive regulation of protein serine/threonine kinase activity / negative regulation of cell growth / kinase binding / autophagy / Z disc / positive regulation of protein localization to nucleus / p53 binding / glucose homeostasis / T cell receptor signaling pathway / spermatogenesis / peptidyl-serine phosphorylation / secretory granule lumen / Regulation of TP53 Activity through Phosphorylation / ficolin-1-rich granule lumen / protein autophosphorylation / regulation of cell cycle / non-specific serine/threonine protein kinase / intracellular signal transduction / negative regulation of cell population proliferation / protein phosphorylation / protein serine kinase activity / protein serine/threonine kinase activity / DNA damage response / Neutrophil degranulation / protein-containing complex binding / magnesium ion binding / signal transduction / mitochondrion / extracellular exosome / extracellular region / nucleoplasm / ATP binding / membrane / nucleus / cytoplasm / cytosol 類似検索 - 分子機能 | ||||||
生物種 | Homo sapiens (ヒト) | ||||||
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: 2.86 Å | ||||||
データ登録者 | Chan, L.M. / Courteau, B.J. / Verba, K.A. | ||||||
資金援助 | 1件
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引用 | ジャーナル: J Struct Biol / 年: 2024 タイトル: High-resolution single-particle imaging at 100-200 keV with the Gatan Alpine direct electron detector. 著者: Lieza M Chan / Brandon J Courteau / Allison Maker / Mengyu Wu / Benjamin Basanta / Hev Mehmood / David Bulkley / David Joyce / Brian C Lee / Stephen Mick / Cory Czarnik / Sahil Gulati / ...著者: Lieza M Chan / Brandon J Courteau / Allison Maker / Mengyu Wu / Benjamin Basanta / Hev Mehmood / David Bulkley / David Joyce / Brian C Lee / Stephen Mick / Cory Czarnik / Sahil Gulati / Gabriel C Lander / Kliment A Verba / 要旨: Developments in direct electron detector technology have played a pivotal role in enabling high-resolution structural studies by cryo-EM at 200 and 300 keV. Yet, theory and recent experiments ...Developments in direct electron detector technology have played a pivotal role in enabling high-resolution structural studies by cryo-EM at 200 and 300 keV. Yet, theory and recent experiments indicate advantages to imaging at 100 keV, energies for which the current detectors have not been optimized. In this study, we evaluated the Gatan Alpine detector, designed for operation at 100 and 200 keV. Compared to the Gatan K3, Alpine demonstrated a significant DQE improvement at these energies, specifically a ∼ 4-fold improvement at Nyquist at 100 keV. In single-particle cryo-EM experiments, Alpine datasets yielded better than 2 Å resolution reconstructions of apoferritin at 120 and 200 keV on a ThermoFisher Scientific (TFS) Glacios microscope fitted with a non-standard SP-Twin lens. We also achieved a ∼ 3.2 Å resolution reconstruction of a 115 kDa asymmetric protein complex, proving Alpine's effectiveness with complex biological samples. In-depth analysis revealed that Alpine reconstructions are comparable to K3 reconstructions at 200 keV, and remarkably, reconstruction from Alpine at 120 keV on a TFS Glacios surpassed all but the 300 keV data from a TFS Titan Krios with GIF/K3. Additionally, we show Alpine's capability for high-resolution data acquisition and screening on lower-end systems by obtaining ∼ 3 Å resolution reconstructions of apoferritin and aldolase at 100 keV and detailed 2D averages of a 55 kDa sample using a side-entry cryo holder. Overall, we show that Gatan Alpine performs well with the standard 200 keV imaging systems and may potentially capture the benefits of lower accelerating voltages, bringing smaller sized particles within the scope of cryo-EM. | ||||||
履歴 |
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-構造の表示
構造ビューア | 分子: MolmilJmol/JSmol |
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-ダウンロードとリンク
-ダウンロード
PDBx/mmCIF形式 | 8vsu.cif.gz | 334.1 KB | 表示 | PDBx/mmCIF形式 |
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PDB形式 | pdb8vsu.ent.gz | 266.1 KB | 表示 | PDB形式 |
PDBx/mmJSON形式 | 8vsu.json.gz | ツリー表示 | PDBx/mmJSON形式 | |
その他 | その他のダウンロード |
-検証レポート
文書・要旨 | 8vsu_validation.pdf.gz | 1.3 MB | 表示 | wwPDB検証レポート |
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文書・詳細版 | 8vsu_full_validation.pdf.gz | 1.3 MB | 表示 | |
XML形式データ | 8vsu_validation.xml.gz | 38.9 KB | 表示 | |
CIF形式データ | 8vsu_validation.cif.gz | 58.4 KB | 表示 | |
アーカイブディレクトリ | https://data.pdbj.org/pub/pdb/validation_reports/vs/8vsu ftp://data.pdbj.org/pub/pdb/validation_reports/vs/8vsu | HTTPS FTP |
-関連構造データ
関連構造データ | 43506MC 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 (文献) |
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類似構造データ | 類似検索 - 機能・相同性F&H 検索 |
-リンク
-集合体
登録構造単位 |
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1 |
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-要素
#1: タンパク質 | 分子量: 41997.109 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) Homo sapiens (ヒト) / 遺伝子: CAB39, MO25, CGI-66 / 細胞株 (発現宿主): expi293T / 発現宿主: Homo sapiens (ヒト) / 参照: UniProt: Q9Y376 |
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#2: タンパク質 | 分子量: 50649.785 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) Homo sapiens (ヒト) / 遺伝子: STK11, LKB1, PJS / 細胞株 (発現宿主): expi293T / 発現宿主: Homo sapiens (ヒト) 参照: UniProt: Q15831, non-specific serine/threonine protein kinase |
#3: タンパク質 | 分子量: 47861.875 Da / 分子数: 1 / 由来タイプ: 組換発現 / 由来: (組換発現) Homo sapiens (ヒト) / 遺伝子: STRADA, LYK5, STRAD / 細胞株 (発現宿主): expi293T / 発現宿主: Homo sapiens (ヒト) / 参照: UniProt: Q7RTN6 |
#4: 化合物 | ChemComp-ADP / |
研究の焦点であるリガンドがあるか | Y |
-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 |
-試料調製
構成要素 | 名称: Heterotrimeric complex of serine/threonine kinase LKB1 with psuedokinase STRADa and scaffolding MO25a タイプ: COMPLEX / Entity ID: #1-#3 / 由来: RECOMBINANT | |||||||||||||||||||||||||
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分子量 | 値: 0.1405 MDa / 実験値: NO | |||||||||||||||||||||||||
由来(天然) | 生物種: Homo sapiens (ヒト) | |||||||||||||||||||||||||
由来(組換発現) | 生物種: Homo sapiens (ヒト) | |||||||||||||||||||||||||
緩衝液 | pH: 7.6 | |||||||||||||||||||||||||
緩衝液成分 |
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試料 | 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES | |||||||||||||||||||||||||
試料支持 | グリッドの材料: GOLD / グリッドのサイズ: 400 divisions/in. / グリッドのタイプ: Quantifoil R1.2/1.3 | |||||||||||||||||||||||||
急速凍結 | 装置: FEI VITROBOT MARK III / 凍結剤: ETHANE / 湿度: 100 % / 凍結前の試料温度: 277 K |
-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: TFS KRIOS |
電子銃 | 電子線源: FIELD EMISSION GUN / 加速電圧: 300 kV / 照射モード: FLOOD BEAM |
電子レンズ | モード: BRIGHT FIELD / 倍率(公称値): 105000 X / 最大 デフォーカス(公称値): 2000 nm / 最小 デフォーカス(公称値): 1000 nm / Cs: 2.7 mm / C2レンズ絞り径: 70 µm |
試料ホルダ | 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER |
撮影 | 電子線照射量: 45.8 e/Å2 / 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: 1293 |
電子光学装置 | エネルギーフィルター名称: GIF Bioquantum / エネルギーフィルタースリット幅: 20 eV |
-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: 979927 | ||||||||||||||||||||||||
3次元再構成 | 解像度: 2.86 Å / 解像度の算出法: FSC 0.143 CUT-OFF / 粒子像の数: 124780 / 対称性のタイプ: POINT | ||||||||||||||||||||||||
原子モデル構築 |
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原子モデル構築 |
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拘束条件 |
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