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基本情報
登録情報 | データベース: PDB / ID: 5fl2 | ||||||
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タイトル | Revisited cryo-EM structure of Inducible lysine decarboxylase complexed with LARA domain of RavA ATPase | ||||||
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![]() | HYDROLASE/ISOMERASE / HYDROLASE-ISOMERASE COMPLEX / ACID-STRESS / ATPASE / RAVA | ||||||
機能・相同性 | ![]() lysine decarboxylase / lysine catabolic process / lysine decarboxylase activity / 加水分解酵素; 酸無水物に作用; 酸無水物に作用・物質の膜輸送を触媒する / guanosine tetraphosphate binding / ATP hydrolysis activity / ATP binding / identical protein binding / cytoplasm / cytosol 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: 6.2 Å | ||||||
![]() | Kandiah, E. / Carriel, D. / Perard, J. / Malet, H. / Bacia, M. / Liu, K. / Chan, S.W.S. / Houry, W.A. / Ollagnier de Choudens, S. / Elsen, S. / Gutsche, I. | ||||||
![]() | ![]() タイトル: Structural insights into the Escherichia coli lysine decarboxylases and molecular determinants of interaction with the AAA+ ATPase RavA. 著者: Eaazhisai Kandiah / Diego Carriel / Julien Perard / Hélène Malet / Maria Bacia / Kaiyin Liu / Sze W S Chan / Walid A Houry / Sandrine Ollagnier de Choudens / Sylvie Elsen / Irina Gutsche / ![]() ![]() 要旨: The inducible lysine decarboxylase LdcI is an important enterobacterial acid stress response enzyme whereas LdcC is its close paralogue thought to play mainly a metabolic role. A unique ...The inducible lysine decarboxylase LdcI is an important enterobacterial acid stress response enzyme whereas LdcC is its close paralogue thought to play mainly a metabolic role. A unique macromolecular cage formed by two decamers of the Escherichia coli LdcI and five hexamers of the AAA+ ATPase RavA was shown to counteract acid stress under starvation. Previously, we proposed a pseudoatomic model of the LdcI-RavA cage based on its cryo-electron microscopy map and crystal structures of an inactive LdcI decamer and a RavA monomer. We now present cryo-electron microscopy 3D reconstructions of the E. coli LdcI and LdcC, and an improved map of the LdcI bound to the LARA domain of RavA, at pH optimal for their enzymatic activity. Comparison with each other and with available structures uncovers differences between LdcI and LdcC explaining why only the acid stress response enzyme is capable of binding RavA. We identify interdomain movements associated with the pH-dependent enzyme activation and with the RavA binding. Multiple sequence alignment coupled to a phylogenetic analysis reveals that certain enterobacteria exert evolutionary pressure on the lysine decarboxylase towards the cage-like assembly with RavA, implying that this complex may have an important function under particular stress conditions. #1: ![]() タイトル: Assembly principles of a unique cage formed by hexameric and decameric E. coli proteins. 著者: Hélène Malet / Kaiyin Liu / Majida El Bakkouri / Sze Wah Samuel Chan / Gregory Effantin / Maria Bacia / Walid A Houry / Irina Gutsche / ![]() ![]() 要旨: A 3.3 MDa macromolecular cage between two Escherichia coli proteins with seemingly incompatible symmetries-the hexameric AAA+ ATPase RavA and the decameric inducible lysine decarboxylase LdcI-is ...A 3.3 MDa macromolecular cage between two Escherichia coli proteins with seemingly incompatible symmetries-the hexameric AAA+ ATPase RavA and the decameric inducible lysine decarboxylase LdcI-is reconstructed by cryo-electron microscopy to 11 Å resolution. Combined with a 7.5 Å resolution reconstruction of the minimal complex between LdcI and the LdcI-binding domain of RavA, and the previously solved crystal structures of the individual components, this work enables to build a reliable pseudoatomic model of this unusual architecture and to identify conformational rearrangements and specific elements essential for complex formation. The design of the cage created via lateral interactions between five RavA rings is unique for the diverse AAA+ ATPase superfamily. | ||||||
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構造ビューア | 分子: ![]() ![]() |
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-関連構造データ
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集合体
登録構造単位 | ![]()
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要素
#1: タンパク質 | 分子量: 80882.453 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: P0A9H3, lysine decarboxylase |
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#2: タンパク質 | 分子量: 11662.463 Da / 分子数: 1 / Fragment: LARA DOMAIN OF RAVA ATPASE / 由来タイプ: 組換発現 / 由来: (組換発現) ![]() ![]() ![]() ![]() 参照: UniProt: P31473, 加水分解酵素; 酸無水物に作用; 酸無水物に作用・物質の膜輸送を触媒する |
-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 between a decamer of inducible lysine decarboxylase LdcI and ten LARA domains of the AAA+ ATPase RavA タイプ: COMPLEX |
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緩衝液 | 名称: 50MM MES PH 6.5, 100MM NACL, 0.2MM PLP, 1MM DTT, 0.01% GLUTARALDEHYDE pH: 6.5 |
試料 | 濃度: 0.6 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES |
試料支持 | 詳細: HOLEY CARBON |
急速凍結 | 詳細: VITRIFICATION 1 -- CRYOGEN- ETHANE, HUMIDITY- 100, TEMPERATURE- 91, INSTRUMENT- FEI VITROBOT MARK III, METHOD- BLOT FOR 2 SECONDS BEFORE PLUNGING, |
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電子顕微鏡撮影
実験機器 | ![]() モデル: Tecnai F30 / 画像提供: FEI Company |
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顕微鏡 | モデル: FEI TECNAI F30 / 日付: 2012年9月14日 / 詳細: AUTOMATIC DATA ACQUISITION WITH FEI EPU SOFTWARE |
電子銃 | 電子線源: ![]() |
電子レンズ | モード: BRIGHT FIELD / 倍率(公称値): 51660 X / 倍率(補正後): 51660 X / 最大 デフォーカス(公称値): 2700 nm / 最小 デフォーカス(公称値): 1500 nm / Cs: 2 mm |
試料ホルダ | 温度: 91 K |
撮影 | 電子線照射量: 20 e/Å2 フィルム・検出器のモデル: GATAN ULTRASCAN 4000 (4k x 4k) |
画像スキャン | デジタル画像の数: 911 |
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解析
CTF補正 | 詳細: INDIVIDUAL PARTICLE WITH FULL CTF CORRECTION AFTER FIRST PEAK | ||||||||||||
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対称性 | 点対称性: D5 (2回x5回 2面回転対称) | ||||||||||||
3次元再構成 | 手法: PROJECTION MATCHING / 解像度: 6.2 Å / ピクセルサイズ(公称値): 1.46466 Å / ピクセルサイズ(実測値): 1.46466 Å 詳細: SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-3206.(DEPOSITION ID: 13911). | ||||||||||||
原子モデル構築 | 手法: FLEXIBLE / プロトコル: FLEXIBLE FIT / 空間: REAL / Target criteria: Cross-correlation coefficient / 詳細: X-RAY | ||||||||||||
原子モデル構築 | PDB-ID: 3N75 Accession code: 3N75 / Source name: PDB / タイプ: experimental model | ||||||||||||
精密化 | 最高解像度: 6.2 Å | ||||||||||||
精密化ステップ | サイクル: LAST / 最高解像度: 6.2 Å
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