+データを開く
-基本情報
登録情報 | データベース: EMDB / ID: EMD-6633 | |||||||||
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タイトル | Glutamate dehydrogenase in complex with NADH and GTP, closed conformation | |||||||||
マップデータ | Reconstruction of TERNARY complex of bovine glutamate dehydrogenase with GTP and NADH. This entry is the CLOSED conformation. | |||||||||
試料 |
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キーワード | enzyme / glutamate metabolism / mitochondria | |||||||||
機能・相同性 | 機能・相同性情報 glutamate dehydrogenase [NAD(P)+] activity / glutamate catabolic process / tricarboxylic acid metabolic process / glutamate dehydrogenase [NAD(P)+] / : / glutamate dehydrogenase (NADP+) activity / glutamate dehydrogenase (NAD+) activity / amino acid metabolic process / glutamine metabolic process / mitochondrial inner membrane ...glutamate dehydrogenase [NAD(P)+] activity / glutamate catabolic process / tricarboxylic acid metabolic process / glutamate dehydrogenase [NAD(P)+] / : / glutamate dehydrogenase (NADP+) activity / glutamate dehydrogenase (NAD+) activity / amino acid metabolic process / glutamine metabolic process / mitochondrial inner membrane / oxidoreductase activity / mitochondrial matrix / nucleotide binding / GTP binding / endoplasmic reticulum / mitochondrion / ATP binding / identical protein binding 類似検索 - 分子機能 | |||||||||
生物種 | Bos taurus (ウシ) / unidentified (未定義) | |||||||||
手法 | 単粒子再構成法 / クライオ電子顕微鏡法 / 解像度: 3.4 Å | |||||||||
データ登録者 | Borgnia MJ / Banerjee S / Merk A / Matthies D / Bartesaghi A / Rao P / Pierson J / Earl LA / Falconieri V / Subramaniam S / Milne JLS | |||||||||
引用 | ジャーナル: Mol Pharmacol / 年: 2016 タイトル: Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase. 著者: Mario J Borgnia / Soojay Banerjee / Alan Merk / Doreen Matthies / Alberto Bartesaghi / Prashant Rao / Jason Pierson / Lesley A Earl / Veronica Falconieri / Sriram Subramaniam / Jacqueline L S Milne / 要旨: Cryo-electron microscopy (cryo-EM) methods are now being used to determine structures at near-atomic resolution and have great promise in molecular pharmacology, especially in the context of mapping ...Cryo-electron microscopy (cryo-EM) methods are now being used to determine structures at near-atomic resolution and have great promise in molecular pharmacology, especially in the context of mapping the binding of small-molecule ligands to protein complexes that display conformational flexibility. We illustrate this here using glutamate dehydrogenase (GDH), a 336-kDa metabolic enzyme that catalyzes the oxidative deamination of glutamate. Dysregulation of GDH leads to a variety of metabolic and neurologic disorders. Here, we report near-atomic resolution cryo-EM structures, at resolutions ranging from 3.2 Å to 3.6 Å for GDH complexes, including complexes for which crystal structures are not available. We show that the binding of the coenzyme NADH alone or in concert with GTP results in a binary mixture in which the enzyme is in either an "open" or "closed" state. Whereas the structure of NADH in the active site is similar between the open and closed states, it is unexpectedly different at the regulatory site. Our studies thus demonstrate that even in instances when there is considerable structural information available from X-ray crystallography, cryo-EM methods can provide useful complementary insights into regulatory mechanisms for dynamic protein complexes. | |||||||||
履歴 |
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-構造の表示
ムービー |
ムービービューア |
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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_6633.map.gz | 59.4 MB | EMDBマップデータ形式 | |
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ヘッダ (付随情報) | emd-6633-v30.xml emd-6633.xml | 15.2 KB 15.2 KB | 表示 表示 | EMDBヘッダ |
画像 | 400_6633.gif 80_6633.gif | 43.2 KB 2.8 KB | ||
アーカイブディレクトリ | http://ftp.pdbj.org/pub/emdb/structures/EMD-6633 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-6633 | HTTPS FTP |
-検証レポート
文書・要旨 | emd_6633_validation.pdf.gz | 398 KB | 表示 | EMDB検証レポート |
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文書・詳細版 | emd_6633_full_validation.pdf.gz | 397.6 KB | 表示 | |
XML形式データ | emd_6633_validation.xml.gz | 6.1 KB | 表示 | |
アーカイブディレクトリ | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6633 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6633 | HTTPS FTP |
-関連構造データ
関連構造データ | 3jd4MC 6630C 6631C 6632C 6634C 6635C 3jczC 3jd0C 3jd1C 3jd2C 3jd3C M: このマップから作成された原子モデル C: 同じ文献を引用 (文献) |
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類似構造データ |
-リンク
EMDBのページ | EMDB (EBI/PDBe) / EMDataResource |
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-マップ
ファイル | ダウンロード / ファイル: emd_6633.map.gz / 形式: CCP4 / 大きさ: 62.5 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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注釈 | Reconstruction of TERNARY complex of bovine glutamate dehydrogenase with GTP and NADH. This entry is the CLOSED conformation. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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: 0.63754 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
密度 |
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対称性 | 空間群: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
詳細 | EMDB XML:
CCP4マップ ヘッダ情報:
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-添付データ
-試料の構成要素
-全体 : Ternary complex of bovine glutamate dehydrogenase with GTP and NADH
全体 | 名称: Ternary complex of bovine glutamate dehydrogenase with GTP and NADH |
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要素 |
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-超分子 #1000: Ternary complex of bovine glutamate dehydrogenase with GTP and NADH
超分子 | 名称: Ternary complex of bovine glutamate dehydrogenase with GTP and NADH タイプ: sample / ID: 1000 詳細: The sample was largely monodisperse. Some chains of hexamers were observed. Image processing revealed the presence of two conformational states (see related entry EMD-6634). 集合状態: one homohexamer of GDH binds 12 molecules of NADH and 6 molecules of GTP Number unique components: 3 |
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分子量 | 理論値: 347 KDa |
-分子 #1: L-glutamate:NAD(P)+ oxidoreductase (deaminating)
分子 | 名称: L-glutamate:NAD(P)+ oxidoreductase (deaminating) / タイプ: protein_or_peptide / ID: 1 Name.synonym: Glutamate dehydrogenase 1, mitochondrial, glutamate dehydrogenase [NAD(P)+], GDH 詳細: Bovine glutamate dehydrogenase (catalog no. G2626; Sigma-Aldrich) was dialyzed overnight against buffer (100 mM potassium phosphate, pH 6.8) prior to fractionation by size-exclusion ...詳細: Bovine glutamate dehydrogenase (catalog no. G2626; Sigma-Aldrich) was dialyzed overnight against buffer (100 mM potassium phosphate, pH 6.8) prior to fractionation by size-exclusion chromatography using a Superdex 200 10/30 column. コピー数: 6 / 集合状態: hexamer / 組換発現: No / データベース: NCBI |
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由来(天然) | 生物種: Bos taurus (ウシ) / 別称: Cow / 組織: Liver / Organelle: Mitochondria / 細胞中の位置: Mitochondrial matrix |
分子量 | 理論値: 56 KDa |
配列 | UniProtKB: Glutamate dehydrogenase 1, mitochondrial GO: nucleotide binding, glutamate dehydrogenase (NAD+) activity, glutamate dehydrogenase [NAD(P)+] activity, glutamate dehydrogenase [NAD(P)+] activity, ATP binding, GTP binding, mitochondrion, ...GO: nucleotide binding, glutamate dehydrogenase (NAD+) activity, glutamate dehydrogenase [NAD(P)+] activity, glutamate dehydrogenase [NAD(P)+] activity, ATP binding, GTP binding, mitochondrion, mitochondrion, mitochondrial inner membrane, mitochondrial matrix, amino acid metabolic process, glutamate catabolic process, glutamine metabolic process, oxidoreductase activity, oxidoreductase activity, GO: 0055114, GO: 0055114, tricarboxylic acid metabolic process InterPro: Glutamate/phenylalanine/leucine/valine dehydrogenase, Glutamate/phenylalanine/leucine/valine dehydrogenase, C-terminal, Glutamate/phenylalanine/leucine/valine dehydrogenase, dimerisation ...InterPro: Glutamate/phenylalanine/leucine/valine dehydrogenase, Glutamate/phenylalanine/leucine/valine dehydrogenase, C-terminal, Glutamate/phenylalanine/leucine/valine dehydrogenase, dimerisation domain, NAD(P)-binding domain |
-分子 #2: [[(2R,3S,4R,5R)-5-(2-amino-6-oxo-3H-purin-9-yl)-3,4-dihydroxyoxol...
分子 | 名称: [[(2R,3S,4R,5R)-5-(2-amino-6-oxo-3H-purin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate タイプ: ligand / ID: 2 / Name.synonym: GTP, Guanosine-5'-triphosphate / コピー数: 6 / 集合状態: monomer / 組換発現: No / データベース: NCBI |
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由来(天然) | 生物種: unidentified (未定義) |
分子量 | 理論値: 1 KDa |
-分子 #3: [[(2R,3S,4R,5R)-5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]me...
分子 | 名称: [[(2R,3S,4R,5R)-5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2R,3S,4R,5R)-5-(3-carbamoyl-4H-pyridin-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl hydrogen phosphate タイプ: ligand / ID: 3 / Name.synonym: NADH, Nicotinamide Adenine Dinucleotide / コピー数: 12 / 集合状態: monomer / 組換発現: No / データベース: NCBI |
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由来(天然) | 生物種: unidentified (未定義) |
分子量 | 理論値: 1 KDa |
-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 | particle |
-試料調製
濃度 | 2.0 mg/mL |
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緩衝液 | pH: 6.8 詳細: 100 mM potassium phosphate, 0.1% n-octyl glucopyranoside, 20 mM GTP, 20 mM NADH |
グリッド | 詳細: 200 mesh Quantifoil R2/2 grids (Quantifoil Micro Tools) |
凍結 | 凍結剤: ETHANE / チャンバー内湿度: 90 % / チャンバー内温度: 90 K / 装置: FEI VITROBOT MARK IV / 手法: Blot for 3-6 seconds before plunging. |
-電子顕微鏡法
顕微鏡 | FEI TITAN KRIOS |
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特殊光学系 | エネルギーフィルター - 名称: Gatan GIF Quantum エネルギーフィルター - エネルギー下限: 0.0 eV エネルギーフィルター - エネルギー上限: 20.0 eV |
日付 | 2014年7月27日 |
撮影 | カテゴリ: CCD フィルム・検出器のモデル: GATAN K2 SUMMIT (4k x 4k) 実像数: 1265 / 平均電子線量: 45 e/Å2 詳細: Every image is the average of 38 frames recorded by the direct electron detector. |
電子線 | 加速電圧: 300 kV / 電子線源: FIELD EMISSION GUN |
電子光学系 | 倍率(補正後): 78426 / 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): 5.0 µm / 最小 デフォーカス(公称値): 1.0 µm |
試料ステージ | 試料ホルダーモデル: FEI TITAN KRIOS AUTOGRID HOLDER |
実験機器 | モデル: Titan Krios / 画像提供: FEI Company |
-画像解析
CTF補正 | 詳細: Each micrograph |
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最終 再構成 | 解像度のタイプ: BY AUTHOR / 解像度: 3.4 Å / 解像度の算出法: OTHER / ソフトウェア - 名称: CTFFIND3, EMAN2, Relion / 使用した粒子像数: 20429 |