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      Cryo-EM reconstruction of AlfA from Bacillus subtilis reveals the structure of a simplified actin-like filament at 3.4 Å resolution

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          Abstract

          Low copy-number plasmid pLS32 of Bacillus subtilis subsp. natto contains a partitioning system that ensures segregation of plasmid copies during cell division. The partitioning locus comprises actin-like protein AlfA, adaptor protein AlfB and the centromeric sequence parN . Similar to the ParMRC partitioning system from E. coli plasmid R1, AlfA filaments form actin-like double helical filaments that arrange into an antiparallel bipolar spindle, which attaches its growing ends to sister plasmids, through interactions with AlfB and parN . Since, compared with ParM and other actin-like proteins, AlfA is highly diverged in sequence, we determined the atomic structure of non-bundling AlfA filaments to 3.4 Å resolution by cryo-EM. The structure reveals how the deletion of subdomain IIB of the canonical actin-fold has been accommodated by unique longitudinal and lateral contacts, whilst still enabling formation of left-handed, double helical, polar and staggered filaments that are architecturally similar to ParM. Through cryo-EM reconstruction of bundling AlfA filaments we obtained a pseudo-atomic model of AlfA doublets: the assembly of two filaments. The filaments are antiparallel, as required by the segregation mechanism, and exactly anti-phasic with 8-fold integer helical symmetry, to enable efficient doublet formation. The structure of AlfA filaments and doublets shows, in atomic detail, signs of the strong evolutionary pressure for simplicity, placed on plasmids: deletion of an entire domain of the actin fold is compensated by changes to all interfaces so that the required properties of polymerisation, nucleotide hydrolysis and antiparallel doublet formation are retained to fulfil the system's biological raison d'être.

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          Author and article information

          Journal
          bioRxiv
          September 19 2017
          Article
          10.1101/190744
          653b6c4b-b6b1-497d-97c0-bdaf2ac4a2ce
          © 2017
          History

          Microbiology & Virology
          Microbiology & Virology

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