Extended Data Fig. 1: Phage protection assays, inputs for IPs, and CD-NTase antibody verification along with CryoEM information.

(a) Image of double agar overlay phage infection assay used to measure efficiency of plating for a lysate of phage T2. E. coli MG1655 expressing the indicated vectors is shown. Zones of clearance (plaques) represent successful phage infection and replication. Apparent plaque forming units (PFU) per mL is calculated for the lysate infecting each bacterial genotype. Fold protection is the PFU per mL of empty vector divided by Vc CBASS, ~10⁴ in this assay. (b) Efficiency of plating of the indicated phage when infecting E. coli expressing CBASS with the indicated genotype. Data plotted as in Fig. 1b. C.D. CD-NTase: DID131AIA.; C.D. capV: C62A. (c) Efficiency of plating of the indicated phage when infecting E. coli expressing V. cholerae CBASS with the indicated genotypes. Data plotted as in Fig. 1b. (d) Western blot analysis of cell lysates (inputs) and αVSV-G immunoprecipitation of E. coli expressing CBASS with the indicated genotypes. These samples correspond to the mass spectrometry in Fig. 1c. αRNAP western blot serves as a loading control for bacterial cells. (-): CBASS operon, CD-NTase without VSV-G; (+): CBASS operon, CD-NTase with N-terminal VSV-G. (e) Whole cell western blot analysis of E. coli expressing either an empty vector (EV) or CBASS (wild-type). αCD-NTase Western blot used a custom CD-NTase antibody; arrow indicates monomeric CD-NTase at the expected molecular weight. αRNAP western blot serves as a loading control for bacterial cells. (f) Efficiency of plating of the indicated phage when infecting E. coli expressing CBASS with the indicated genotypes. Data plotted as in Fig. 1b. (g) Whole cell western blot analysis of E. coli expressing the indicated genotypes of CBASS. Data are the input for the immunoprecipitation presented in Fig. 1d. (h) Whole cell western blot analysis of E. coli expressing the indicated genotypes of CBASS. Data are the input for the immunoprecipitation presented in Fig. 1e. For (f),(g) and (h) ±ϕ indicates phage T2 at an MOI of 2. (i) Operon structure of CBASS from E. cloacae. See Supplementary Table 5 for relevant accession numbers. (j) Size exclusion chromatography elution profile (Superdex 200 Increase 10/300 GL) and SDS-PAGE analysis of E. cloacae Cap2–CD-NTase. The fraction used for cryoEM analysis is shaded in gray. C.D. Cap2: C109A/C548A. (k) Representative electron micrograph of E. cloacae Cap2–CD-NTase. (l) Fourier Shell Correlation (FSC) curve for the final refinement of the 2:2 Cap2–CD-NTase complex. (m) 3D FSC analysis⁵⁵ for the 2:2 Cap2–CD-NTase complex. (n) Fourier Shell Correlation (FSC) curve for the final refinement of the 2:1 Cap2–CD-NTase complex. (o) 3D FSC analysis for the 2:1 Cap2–CD-NTase complex. (p) Local resolution of the final refined map for the 2:2 Cap2–CD-NTase complex, colored from blue (≤1.8 Å) to magenta (≥4.00 Å). (q) Local resolution of the final refined map for the 2:1 Cap2–CD-NTase complex colored from blue (≤1.8 Å) to magenta (≥4.00 Å). Outline indicates the areas of missing density compared to the 2:2 Cap2–CD-NTase complex.