French Infrastructure for Integrated Structural Biology
ANR-10-INSB-05-02
フランス
Grenoble Instruct-ERIC Center
UMS 3518 CNRS-CEA-UJF-EMBL
フランス
European Molecular Biology Organization
ALTF441-2017
フランス
引用
ジャーナル: Commun Biol / 年: 2020 タイトル: Structural insights into ATP hydrolysis by the MoxR ATPase RavA and the LdcI-RavA cage-like complex. 著者: Matthew Jessop / Benoit Arragain / Roger Miras / Angélique Fraudeau / Karine Huard / Maria Bacia-Verloop / Patrice Catty / Jan Felix / Hélène Malet / Irina Gutsche / 要旨: The hexameric MoxR AAA+ ATPase RavA and the decameric lysine decarboxylase LdcI form a 3.3 MDa cage, proposed to assist assembly of specific respiratory complexes in E. coli. Here, we show that ...The hexameric MoxR AAA+ ATPase RavA and the decameric lysine decarboxylase LdcI form a 3.3 MDa cage, proposed to assist assembly of specific respiratory complexes in E. coli. Here, we show that inside the LdcI-RavA cage, RavA hexamers adopt an asymmetric spiral conformation in which the nucleotide-free seam is constrained to two opposite orientations. Cryo-EM reconstructions of free RavA reveal two co-existing structural states: an asymmetric spiral, and a flat C2-symmetric closed ring characterised by two nucleotide-free seams. The closed ring RavA state bears close structural similarity to the pseudo two-fold symmetric crystal structure of the AAA+ unfoldase ClpX, suggesting a common ATPase mechanism. Based on these structures, and in light of the current knowledge regarding AAA+ ATPases, we propose different scenarios for the ATP hydrolysis cycle of free RavA and the LdcI-RavA cage-like complex, and extend the comparison to other AAA+ ATPases of clade 7.
pH: 7.9 詳細: 20 mM Tris pH 7.9, 300 mM NaCl, 2 mM ADP, 10 mM MgCl 2 , 0.1 mM PLP and 1 mM DTT
凍結
凍結剤: ETHANE
詳細
Concentrations of LdcI and RavA were 0.38 mg/ml (4.67 microM) and 0.6 mg/ml (10.64 microM) respectively.
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電子顕微鏡法
顕微鏡
FEI POLARA 300
詳細
Data collection was performed on an FEI Polara microscope operated at 300 kV. Movies of 40 frames were collected with a total exposure time of 8s and a total dose of 40e-/Angstrom^2 on a K2 summit direct electron detector (Gatan) at a magnification of 41270x, corresponding to 1.21 Angstrom/pixel at the specimen level.
倍率(補正後): 41270 / 照射モード: FLOOD BEAM / 撮影モード: BRIGHT FIELD
実験機器
モデル: Tecnai Polara / 画像提供: FEI Company
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画像解析
粒子像選択
選択した数: 18902 詳細: The cleanded dataset contains 11866 particles. Dataset expansion (C5) resulted in a dataset containing 59330 particles.
CTF補正
ソフトウェア - 名称: Gctf
初期モデル
モデルのタイプ: INSILICO MODEL In silico モデル: The initial 3D model based on 2D class averages was calculated with sxviper (SPARX) (Hohn et al., 2007) imposing D5 symmetry.
Local resolution estimation and subsequent filtering of maps were performed in Relion3.0. For fitting of atomic models in the resulting filtered maps, we used the previously-determined X-ray structures of LdcI (PDB ID: 3N75) (Kanjee et al., 2011) and RavA (PDB ID: 3NBX) (El Bakkouri et al., 2010). In each map, two decameric LdcI molecules extracted from PDB 3N75 and one spiral RavA hexamer extracted from a continuous RavA helix generated from PDB 3NBX were fitted separately using iMODFIT (Lopez-Blanco and Chacon, 2013), followed by a single round of B-factor (ADP) refinement in Phenix.
精密化
プロトコル: FLEXIBLE FIT
得られたモデル
PDB-6q7m: Spiral structure of E. coli RavA in the RavA-LdcI cage-like complex