Fig 1: Evidence for the heterotrimeric stoichiometry of Type III REs. (A) Native mass spectrum of wt EcoP15I. The Y-axis is relative intensity, scaled to the most intense peak in the spectrum, which is the 31+ charge state of the enzyme complex. The numbers above the peaks are the measured charge states for the given molecular species. The observed molecular weight (259.3 kDa) corresponds closely to that expected of the Res1Mod2 heterotrimer (259145 Da, Table 1). (B) SEC-MALS traces of various Type III REs. All enzymes yielded a single elution peak with a molecular weight supporting the Res1Mod2 subunit stoichiometry, except for PstII, which eluted as a double peak indicating a mixture of Mod2 and Res1Mod2 species. Solid lines represent the normalized light scattering (left Y-axis), whereas dotted lines show the calculated molecular masses (right Y-axis). Traces are shown in two separate graphs for clarity, noting that the wt EcoP15I and EcoP15I obtained from NEB had the same elution volume. See “Evidence for a Res1Mod2 stoichiometry for Type III REs” in the Results section and Table 3 for further details.