Extended Data Fig. 2: Oligomeric state analysis and biochemical profiling of KpOptS.

(a) Multiple sequence alignment of representative Panoptes OptS, type-III CRISPR polymerase palm domains, and GGDEF palm domains. Residues important for catalysis are indicated with black asterisks. Orange asterisk indicated nucleobase recognition residues for Kp and VnOptS. (b–e) The KpOptS tetramer contains three possible dimer (pair of protomers) interfaces shown in order of decreasing buried surface area (determined with PISA analysis, see Supplementary Table 1). (f) Size exclusion chromatography reveals a monodisperse signal for KpOptS with an elution volume consistent with an oligomer >29 kDa (monomer = 14 kDa). Grey dots indicate molecular weight standards: Blue dextran, 46.2 mL, 2000 kDa; Ferritin, 59.8 mL, 440 kDa; Aldolase, 71.0 mL, 158 kDa; Conalbumin, 78.9 mL, 75 kDa; Ovalbumin, 85.3 mL, 43 kDa; Carbonic anhydrase, 91.5 mL, 29 kDa; Ribonuclease A, 98.6 mL, 13.7 kDa; Aprotinin, 104.4 mL, 6.5 kDa. The trace is representative of at least two biological replicates. (g, h) Mass photometry results showing distribution of sizes detected for KpOptS in solution at 50 nM. (i) HPLC analysis of mixture of 4×NTPs (ATP, GTP, UTP, CTP) compared with the product of KpOptS when incubated with NTPs. Traces representative of at least three biological replicates. (j) HPLC analysis of GTP compared with the product of KpOptS when incubated with GTP. Traces representative of at least three biological replicates. (k) HPLC analysis of ATP and GTP compared with the product of KpOptS when incubated with ATP and GTP. Traces representative of at least three biological replicates. (l) HPLC analysis of 2′,2′-cGAMP, 2′,3′-cGAMP, 3′,2′-cGAMP, and 3′,3′-cGAMP chemical standards compared with the product of KpOptS incubated with ATP and GTP. Traces representative of at least three biological replicates. (m) P1 nuclease and CIP treatment of KpOptS ATP-only product. Traces representative of at least three biological replicates.

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