Dpni fast digest

All protein-encoding constructs were amplified directly from Streptomyces virginiae genomic DNA using forward and reverse primers incorporating BamHI and HindIII restriction sites, respectively (Supplementary Data 1), and were ligated into the corresponding sites of vector pBG-102 (with the exception of VirC and its quadruple mutant which were cloned into pLM-302). Vector pBG-102 codes for a His₆-SUMO tag and pLM-302 codes for a His₆-maltose binding protein (MBP) tag (Centre for Structural Biology, Vanderbilt University). In both cases, cleavage of the tags resulted in a non-native N-terminal Gly-Pro-Gly-Ser sequence. The sequences of all constructs were verified by DNA sequencing prior to protein expression studies. Site-directed mutations were introduced into ACP_5a and VirD by PCR using mutagenic oligonucleotides (Supplementary Data 1) and Phusion High-Fidelity polymerase, followed by digestion of the parental DNA by 1 μL of DpnI Fast digest (Thermo Fischer Scientific). The presence of the correct mutations was confirmed by sequencing.

Plasmid pGEX-LcsJ (a kind gift of Professor P.F. Leadlay; lcsJ is equivalent to lkcE^{23 (link)}) was digested with BamHI and XhoI, and the resulting fragment cloned into the equivalent sites of plasmid pBG-102 (Center for Structural Biology, Vanderbilt University), which codes for a His₆-SUMO tag, to yield pBG-102-LkcE (the full list of plasmids used in this study is provided in Supplementary Table 2). The sequence of lkcE was re-verified by sequencing. Site-directed mutations were introduced into lkcE by PCR using mutagenic oligonucleotides (Supplementary Table 1) and Phusion high-fidelity polymerase, followed by digestion of the parental DNA by 1 μL of DpnI Fast digest (Thermo Fischer Scientific). The presence of the correct mutations was confirmed by sequencing.