+データを開く
-基本情報
登録情報 | データベース: EMDB / ID: EMD-25906 | |||||||||||||||
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タイトル | Structure of Leucine Rich Repeat Kinase 2's ROC domain interacting with the microtubule facing the minus end | |||||||||||||||
マップデータ | Sharpened map | |||||||||||||||
試料 |
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キーワード | parkinson's disease / microtubule / kinase / gtpase / CYTOSOLIC PROTEIN | |||||||||||||||
機能・相同性 | 機能・相同性情報 peroxidase inhibitor activity / caveola neck / negative regulation of thioredoxin peroxidase activity by peptidyl-threonine phosphorylation / negative regulation of protein processing involved in protein targeting to mitochondrion / Wnt signalosome assembly / regulation of branching morphogenesis of a nerve / beta-catenin destruction complex binding / regulation of kidney size / regulation of neuron maturation / tangential migration from the subventricular zone to the olfactory bulb ...peroxidase inhibitor activity / caveola neck / negative regulation of thioredoxin peroxidase activity by peptidyl-threonine phosphorylation / negative regulation of protein processing involved in protein targeting to mitochondrion / Wnt signalosome assembly / regulation of branching morphogenesis of a nerve / beta-catenin destruction complex binding / regulation of kidney size / regulation of neuron maturation / tangential migration from the subventricular zone to the olfactory bulb / protein localization to endoplasmic reticulum exit site / GTP-dependent protein kinase activity / regulation of neuroblast proliferation / regulation of ER to Golgi vesicle-mediated transport / negative regulation of late endosome to lysosome transport / regulation of mitochondrial depolarization / negative regulation of protein targeting to mitochondrion / regulation of synaptic vesicle transport / regulation of lysosomal lumen pH / positive regulation of dopamine receptor signaling pathway / amphisome / regulation of CAMKK-AMPK signaling cascade / cytoplasmic side of mitochondrial outer membrane / co-receptor binding / mitochondrion localization / regulation of retrograde transport, endosome to Golgi / negative regulation of excitatory postsynaptic potential / regulation of dopamine receptor signaling pathway / negative regulation of autophagosome assembly / positive regulation of microglial cell activation / neuron projection arborization / positive regulation of synaptic vesicle endocytosis / JUN kinase kinase kinase activity / olfactory bulb development / regulation of protein kinase A signaling / regulation of dendritic spine morphogenesis / striatum development / multivesicular body, internal vesicle / protein localization to mitochondrion / cellular response to dopamine / endoplasmic reticulum organization / positive regulation of protein autoubiquitination / presynaptic cytosol / positive regulation of programmed cell death / GTP metabolic process / Wnt signalosome / regulation of canonical Wnt signaling pathway / negative regulation of protein processing / negative regulation of GTPase activity / syntaxin-1 binding / regulation of reactive oxygen species metabolic process / exploration behavior / protein kinase A binding / regulation of locomotion / regulation of synaptic vesicle exocytosis / PTK6 promotes HIF1A stabilization / clathrin binding / Golgi-associated vesicle / negative regulation of macroautophagy / neuromuscular junction development / lysosome organization / regulation of mitochondrial fission / intracellular distribution of mitochondria / autolysosome / Golgi organization / positive regulation of nitric-oxide synthase biosynthetic process / locomotory exploration behavior / endoplasmic reticulum exit site / microvillus / Rho protein signal transduction / MAP kinase kinase kinase activity / positive regulation of protein kinase activity / canonical Wnt signaling pathway / cellular response to manganese ion / negative regulation of endoplasmic reticulum stress-induced intrinsic apoptotic signaling pathway / positive regulation of autophagy / JNK cascade / phosphorylation / regulation of synaptic transmission, glutamatergic / dendrite cytoplasm / GTPase activator activity / tubulin binding / cellular response to starvation / neuron projection morphogenesis / SNARE binding / regulation of membrane potential / excitatory postsynaptic potential / negative regulation of protein phosphorylation / positive regulation of protein ubiquitination / mitochondrion organization / negative regulation of protein binding / regulation of autophagy / determination of adult lifespan / mitochondrial membrane / peptidyl-threonine phosphorylation / calcium-mediated signaling / positive regulation of MAP kinase activity / trans-Golgi network / regulation of protein stability / terminal bouton 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: 5.2 Å | |||||||||||||||
データ登録者 | Matyszewski M / Leschziner AE | |||||||||||||||
資金援助 | 米国, 4件
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引用 | ジャーナル: Nat Struct Mol Biol / 年: 2022 タイトル: Structural basis for Parkinson's disease-linked LRRK2's binding to microtubules. 著者: David M Snead / Mariusz Matyszewski / Andrea M Dickey / Yu Xuan Lin / Andres E Leschziner / Samara L Reck-Peterson / 要旨: Leucine-rich repeat kinase 2 (LRRK2) is one of the most commonly mutated genes in familial Parkinson's disease (PD). Under some circumstances, LRRK2 co-localizes with microtubules in cells, an ...Leucine-rich repeat kinase 2 (LRRK2) is one of the most commonly mutated genes in familial Parkinson's disease (PD). Under some circumstances, LRRK2 co-localizes with microtubules in cells, an association enhanced by PD mutations. We report a cryo-EM structure of the catalytic half of LRRK2, containing its kinase, in a closed conformation, and GTPase domains, bound to microtubules. We also report a structure of the catalytic half of LRRK1, which is closely related to LRRK2 but is not linked to PD. Although LRRK1's structure is similar to that of LRRK2, we find that LRRK1 does not interact with microtubules. Guided by these structures, we identify amino acids in LRRK2's GTPase that mediate microtubule binding; mutating them disrupts microtubule binding in vitro and in cells, without affecting LRRK2's kinase activity. Our results have implications for the design of therapeutic LRRK2 kinase inhibitors. | |||||||||||||||
履歴 |
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-構造の表示
添付画像 |
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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 | emd_25906.map.gz | 97.1 MB | EMDBマップデータ形式 | |
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ヘッダ (付随情報) | emd-25906-v30.xml emd-25906.xml | 22.1 KB 22.1 KB | 表示 表示 | EMDBヘッダ |
FSC (解像度算出) | emd_25906_fsc.xml | 10.4 KB | 表示 | FSCデータファイル |
画像 | emd_25906.png | 88.1 KB | ||
マスクデータ | emd_25906_msk_1.map | 103 MB | マスクマップ | |
Filedesc metadata | emd-25906.cif.gz | 6.6 KB | ||
その他 | emd_25906_additional_1.map.gz emd_25906_half_map_1.map.gz emd_25906_half_map_2.map.gz | 50.9 MB 95.7 MB 95.7 MB | ||
アーカイブディレクトリ | http://ftp.pdbj.org/pub/emdb/structures/EMD-25906 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-25906 | HTTPS FTP |
-検証レポート
文書・要旨 | emd_25906_validation.pdf.gz | 950.3 KB | 表示 | EMDB検証レポート |
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文書・詳細版 | emd_25906_full_validation.pdf.gz | 949.9 KB | 表示 | |
XML形式データ | emd_25906_validation.xml.gz | 17.9 KB | 表示 | |
CIF形式データ | emd_25906_validation.cif.gz | 23.2 KB | 表示 | |
アーカイブディレクトリ | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-25906 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-25906 | HTTPS FTP |
-関連構造データ
関連構造データ | 7thyMC 7thzC C: 同じ文献を引用 (文献) M: このマップから作成された原子モデル |
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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
EMDBのページ | EMDB (EBI/PDBe) / EMDataResource |
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「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_25906.map.gz / 形式: CCP4 / 大きさ: 103 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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注釈 | Sharpened map | ||||||||||||||||||||||||||||||||||||
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: 1.16 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
-マスク #1
ファイル | emd_25906_msk_1.map | ||||||||||||
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投影像・断面図 |
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密度ヒストグラム |
-追加マップ: Non-sharpened map
ファイル | emd_25906_additional_1.map | ||||||||||||
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注釈 | Non-sharpened map | ||||||||||||
投影像・断面図 |
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密度ヒストグラム |
-ハーフマップ: Half map 1
ファイル | emd_25906_half_map_1.map | ||||||||||||
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注釈 | Half map 1 | ||||||||||||
投影像・断面図 |
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密度ヒストグラム |
-ハーフマップ: Half map 2
ファイル | emd_25906_half_map_2.map | ||||||||||||
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注釈 | Half map 2 | ||||||||||||
投影像・断面図 |
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密度ヒストグラム |
-試料の構成要素
-全体 : LRRK2RCKW filament bound to a 11-pf microtubule with MLi-2 present
全体 | 名称: LRRK2RCKW filament bound to a 11-pf microtubule with MLi-2 present |
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要素 |
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-超分子 #1: LRRK2RCKW filament bound to a 11-pf microtubule with MLi-2 present
超分子 | 名称: LRRK2RCKW filament bound to a 11-pf microtubule with MLi-2 present タイプ: complex / ID: 1 / 親要素: 0 / 含まれる分子: #1 |
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-分子 #1: Leucine-rich repeat serine/threonine-protein kinase 2
分子 | 名称: Leucine-rich repeat serine/threonine-protein kinase 2 タイプ: protein_or_peptide / ID: 1 / コピー数: 1 / 光学異性体: LEVO / EC番号: non-specific serine/threonine protein kinase |
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由来(天然) | 生物種: Homo sapiens (ヒト) |
分子量 | 理論値: 22.191762 KDa |
組換発現 | 生物種: Spodoptera frugiperda (ツマジロクサヨトウ) |
配列 | 文字列: YNRMKLMIVG NTGSGKTTLL QQLMKTKKSD LGMQSATVGI DVKDWPIQIR DKRKRDLVLN VWDFAGREEF YSTHPHFMTQ RALYLAVYD LSKGQAEVDA MKPWLFNIKA RASSSPVILV GTHLDVSDEK QRKACMSKIT KELLNKRGFP AIRDYHFVNA T EESDALAK ...文字列: YNRMKLMIVG NTGSGKTTLL QQLMKTKKSD LGMQSATVGI DVKDWPIQIR DKRKRDLVLN VWDFAGREEF YSTHPHFMTQ RALYLAVYD LSKGQAEVDA MKPWLFNIKA RASSSPVILV GTHLDVSDEK QRKACMSKIT KELLNKRGFP AIRDYHFVNA T EESDALAK LRKTIINESL NFKIRDQLVV GQLIPD UniProtKB: Leucine-rich repeat serine/threonine-protein kinase 2 |
-分子 #2: GUANOSINE-5'-DIPHOSPHATE
分子 | 名称: GUANOSINE-5'-DIPHOSPHATE / タイプ: ligand / ID: 2 / コピー数: 1 / 式: GDP |
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分子量 | 理論値: 443.201 Da |
Chemical component information | ChemComp-GDP: |
-実験情報
-構造解析
手法 | クライオ電子顕微鏡法 |
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解析 | 単粒子再構成法 |
試料の集合状態 | filament |
-試料調製
緩衝液 | pH: 7.4 構成要素:
詳細: This is the final dilution buffer. The incubation buffer consisted of 1x BRB80, 10% glycerol, 1mM DTT, 1mM GTP, 1mM MgCl2, 10 uM taxol, and 5 uM MLi-2. Sample was diluted 3-fold right before ...詳細: This is the final dilution buffer. The incubation buffer consisted of 1x BRB80, 10% glycerol, 1mM DTT, 1mM GTP, 1mM MgCl2, 10 uM taxol, and 5 uM MLi-2. Sample was diluted 3-fold right before freezing with the final buffer. | ||||||||||||||||||
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グリッド | モデル: Homemade / 支持フィルム - 材質: CARBON / 支持フィルム - トポロジー: LACEY / 前処理 - タイプ: GLOW DISCHARGE / 前処理 - 時間: 45 sec. 詳細: Using EMS LC-300 Lacey Carbon grids (Not homemade, EMS not a choice for grid company) | ||||||||||||||||||
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 | ||||||||||||||||||
詳細 | 4.5 uM of LRRK2RCKW was allowed to incubate with 2.25 uM of tubulin dimer, causing both to co-polymerize. 5 uM of MLi-2 was present as well. The sample was diluted 3-fold right before freezing (1.5 uM LRRK2RCKW concentration final). |
-電子顕微鏡法
顕微鏡 | FEI TALOS ARCTICA |
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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 / 撮影したグリッド数: 2 / 平均露光時間: 10.0 sec. / 平均電子線量: 55.0 e/Å2 / 詳細: 250 ms frames |
電子線 | 加速電圧: 200 kV / 電子線源: FIELD EMISSION GUN |
電子光学系 | C2レンズ絞り径: 70.0 µm / 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD / Cs: 2.7 mm / 最大 デフォーカス(公称値): 1.5 µm / 最小 デフォーカス(公称値): 1.5 µm / 倍率(公称値): 36000 |
試料ステージ | ホルダー冷却材: NITROGEN |
実験機器 | モデル: Talos Arctica / 画像提供: FEI Company |
+画像解析
-原子モデル構築 1
詳細 | Used AlphaFold model as initial model (Q5S007) using only the ROC domain. TUB1 was added to the initial refinement to prevent ROC model from entering density reserved for the microtubule. TUB1 was discarded after the initial refinement. |
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精密化 | プロトコル: FLEXIBLE FIT |
得られたモデル | PDB-7thy: |