National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
GM105691
米国
引用
ジャーナル: Nucleic Acids Res / 年: 2023 タイトル: Structure, substrate binding and activity of a unique AAA+ protein: the BrxL phage restriction factor. 著者: Betty W Shen / Lindsey A Doyle / Rachel Werther / Abigail A Westburg / Daniel P Bies / Stephanie I Walter / Yvette A Luyten / Richard D Morgan / Barry L Stoddard / Brett K Kaiser / 要旨: Bacteriophage exclusion ('BREX') systems are multi-protein complexes encoded by a variety of bacteria and archaea that restrict phage by an unknown mechanism. One BREX factor, termed BrxL, has been ...Bacteriophage exclusion ('BREX') systems are multi-protein complexes encoded by a variety of bacteria and archaea that restrict phage by an unknown mechanism. One BREX factor, termed BrxL, has been noted to display sequence similarity to various AAA+ protein factors including Lon protease. In this study we describe multiple CryoEM structures of BrxL that demonstrate it to be a chambered, ATP-dependent DNA binding protein. The largest BrxL assemblage corresponds to a dimer of heptamers in the absence of bound DNA, versus a dimer of hexamers when DNA is bound in its central pore. The protein displays DNA-dependent ATPase activity, and ATP binding promotes assembly of the complex on DNA. Point mutations within several regions of the protein-DNA complex alter one or more in vitro behaviors and activities, including ATPase activity and ATP-dependent association with DNA. However, only the disruption of the ATPase active site fully eliminates phage restriction, indicating that other mutations can still complement BrxL function within the context of an otherwise intact BREX system. BrxL displays significant structural homology to MCM subunits (the replicative helicase in archaea and eukaryotes), implying that it and other BREX factors may collaborate to disrupt initiation of phage DNA replication.
凍結剤: ETHANE / チャンバー内湿度: 95 % / チャンバー内温度: 298 K / 装置: FEI VITROBOT MARK IV
詳細
This sample was mono-dispersed
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電子顕微鏡法
顕微鏡
FEI TALOS ARCTICA
撮影
フィルム・検出器のモデル: DIRECT ELECTRON DE-10 (5k x 4k) 検出モード: COUNTING / 撮影したグリッド数: 2 / 実像数: 3000 / 平均露光時間: 2.0 sec. / 平均電子線量: 50.0 e/Å2 詳細: 2980 movies at 1.12 pixel was extracted at box size of 406 pix, fourier cropped to box size of 392 pix and combined with 100 movies collected at 1.16 pix size
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: OTHER / 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): 1.2 µm / 倍率(公称値): 38000