X-ray diffraction data were recorded at either Diamond Light Source beamline I04-1 (λ = 0.9281 Å), I03 (λ = 0.9763 Å), or I24 or collected in-house, on a Rigaku 007HFM copper (λ = 1.54 Å) rotating anode x-ray generator with a Saturn 944 charge-coupled device (CCD) detector at 100 K. Data were reduced, integrated, and scaled using a toolbox autoPROC (3335), xia2 (3638), or HKL2000(39); OphAΔC6 Y76F-SAM complex data were reindexed to P21212 in CCP4(40) and further merged with Aimless in CCP4 (40). Automated structure solution pipeline CRANK2 (41) was used for experimental phasing using selenium single-wavelength anomalous diffraction (SAD) methods for the Se-OphAΔC18 data set. The model was further improved and refined using COOT (42), REFMAC5 (43), and Phenix (44). All the other structures were solved by molecular replacement with OphAΔC18 as the initial search model using Phaser (45) and refined as described above. Each final model was verified with MolProbity (46). Refinement statistics were summarized in table S1, and electron density in fig. S6. Although the main chain electron density of the substrate peptide was clear, despite the relatively high resolution of the structures, the side chains of the substrate peptides could not be interpreted unambiguously in most cases. We attribute this to disorder within the register, that is, substrate peptide docks in multiple conformations in the crystal, which, although sharing the same backbone orientation, have different side chains. Consequently, although we have modeled a particular register in the structures, our interpretations have avoided placing weight on the identity of the side chain at position i. To obtain the ratio of SAM, SAH, or sinefungin in crystals, structures of Y63F-sinefungin (50 mM), R72A-SAM, Y76F-SAM, Y98A-SAM, and W400A-sinefungin were refined for many rounds by Phenix until occupancy of SAH, SAM, or sinefungin (starting point is 0.5 occupancy of SAM/sinefungin and SAH) no longer change. Electrostatic surfaces were calculated with CCP4MG (47). All crystallographic figures were generated using Pymol (Schrödinger LLC). Coordinates and data have been deposited with the wwPDB (Worldwide Protein Data Bank).

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