Fig. 4: Structural and functional characterization of ZorC and ZorD.
From: Structure and mechanism of the Zorya anti-phage defence system
a, Ribbon model representation of ZorC. Residues from Arg58 to Pro478 were modelled based on EM density. b, Details of the ZorC EH signature motif. c, The effects of ZorC mutations on EcZorI-mediated anti-phage defence, as measured using EOP assays. d, In vitro interaction of EcZorC with 200 nM dsDNA (18 bp, 50% GC content, 5′ FAM-labelled dsDNA; the sequence is shown in Extended Data Fig. 7h), ZorC concentrations were as follows for lanes 1, 3, 4, 5, 6 and 7, respectively: 2,500, 100, 250, 500, 1,000 and 2,500 nM. e, The effects of ZorC mutations on dsDNA-binding activity. All reactions were made to a final concentration of 100 nM of dsDNA and 2,000 nM of protein. f, Representatives of high-resolution two-dimensional classes of apo EcZorC and EcZorC–dsDNA complex images from cryo-EM. Scale bars, 100 Å. g, Ribbon model representation of EcZorD in a complex with ATP-γ-S, with the bound ATP-γ-S shown as a sphere representation. h, Details of ATP-γ-S-binding sites. The backbone of the DEAQ box motif (ZorD residues 730–733) is coloured in magenta. Conserved negatively charged residues surrounding the DEAQ box motif are shown. i, The effects of ZorD mutations on EcZorI-mediated anti-phage defence, as measured using EOP assays. ΔNTD represents ZorD(Δ1–502) and ΔCTD represents ZorD(Δ503–1080). For c and i, data are the mean of at least three replicates (datapoints indicate replicates) and are normalized to the control samples lacking EcZorI. Data for additional phages are provided in Extended Data Fig. 6a. j, ZorD CTD degrades linear plasmid DNA. k, ZorD CTD degrades phage Bas24 genomic DNA (gDNA). Data for additional phages are provided in Extended Data Fig. 8e. l, Time-lapse montage of SYTOX-Orange-labelled Bas24 infections. The arrows indicate phage particles that appear to adsorb and inject their DNA. Scale bars, 4 µm. m, Schematic of the apparent transfer of labelled phage DNA from the capsid to inside the cell. n, Quantification of intracellular fluorescence levels over time in individual E. coli cells, comparing the infection dynamics in Zorya-deficient cells and EcZorI-expressing cells (data from l, plus four additional replicates). The dotted points indicate cell lysis of E. coli cells lacking EcZorI. The bold lines represent the mean estimated from a linear regression analysis. Images in d, e, j and k are representatives of at least three replicates. CTD, C-terminal domain; NTD, N-terminal domain; WT, wild type.
