Bharat Tanmay A M, Murshudov Garib N, Sachse Carsten, Löwe Jan
Structural Studies Division, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg 69117, Germany.
Nature. 2015 Jul 2;523(7558):106-10. doi: 10.1038/nature14356. Epub 2015 Apr 27.
Active segregation of Escherichia coli low-copy-number plasmid R1 involves formation of a bipolar spindle made of left-handed double-helical actin-like ParM filaments. ParR links the filaments with centromeric parC plasmid DNA, while facilitating the addition of subunits to ParM filaments. Growing ParMRC spindles push sister plasmids to the cell poles. Here, using modern electron cryomicroscopy methods, we investigate the structures and arrangements of ParM filaments in vitro and in cells, revealing at near-atomic resolution how subunits and filaments come together to produce the simplest known mitotic machinery. To understand the mechanism of dynamic instability, we determine structures of ParM filaments in different nucleotide states. The structure of filaments bound to the ATP analogue AMPPNP is determined at 4.3 Å resolution and refined. The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions. Also using electron cryomicroscopy, we reconstruct ParM doublets forming antiparallel spindles. Finally, with whole-cell electron cryotomography, we show that doublets are abundant in bacterial cells containing low-copy-number plasmids with the ParMRC locus, leading to an asynchronous model of R1 plasmid segregation.
大肠杆菌低拷贝数质粒R1的主动分离涉及由左手双螺旋肌动蛋白样ParM细丝构成的双极纺锤体的形成。ParR将细丝与着丝粒parC质粒DNA相连，同时促进亚基添加到ParM细丝上。生长中的ParMRC纺锤体将姐妹质粒推向细胞两极。在此，我们使用现代电子冷冻显微镜方法研究了体外和细胞内ParM细丝的结构与排列，以近原子分辨率揭示了亚基和细丝如何聚集在一起形成已知最简单的有丝分裂机制。为了解动态不稳定性的机制，我们确定了处于不同核苷酸状态的ParM细丝的结构。与ATP类似物AMPPNP结合的细丝结构在4.3 Å分辨率下确定并得到优化。ParM细丝结构显示出强纵向界面和较弱的横向相互作用。我们还利用电子冷冻显微镜重建了形成反平行纺锤体的ParM双联体。最后，通过全细胞电子冷冻断层扫描，我们表明双联体在含有具有ParMRC位点的低拷贝数质粒的细菌细胞中大量存在，从而得出R1质粒分离的异步模型。
