Extended Data Fig. 5: Homology models of PAMmla predicted PAM-altering mutations.

a, An E1219Y substitution may facilitate interaction with the amino group of bases in the 3^rd position of the PAM. b, R1335Q permits major groove readout of both bases of a C-G pair in the 3^rd position of the PAM. c, E1219C, R1335M, and T1337V substitutions form a hydrophobic pocket to promote van der Waals interactions with the methyl group of thymine in the 3^rd position of the PAM. Representation of the protein surface is colored by lipophilicity potential. d, T1337R results in direct major groove readout of guanine in the 4^th position of the PAM. e, T1337K facilitates major groove readout of oxygen group of bases in the 4^th position the PAM. f, R1335L and T1337C substitutions form a hydrophobic pocket to promote recognition of thymine in the 4^th position of the PAM. Protein surface is colored by lipophilicity potential. g, D1135L disrupts coordination with R1114, enabling improved flexibility of the R1114 side chain to contact the NTS backbone. WT SpCas9 is overlaid in grey. h, Substitution of G1218 to a positive residue establishes additional non-specific contacts with the NTS backbone. i, S1136W and D1135L result in a shift of the NTS and TS backbone towards the PAM-interacting domain, enabling novel base specific interactions in nearby regions. WT SpCas9 is overlaid in grey. For panels a–i, amino acid and PAM DNA base substitutions were modeled on the structure of SpG (PDB: 8U3Y)⁶ using Coot⁹⁵, except for substitutions T1337R, T1337K, and T1337C which were modeled using SpCas9-VRER (PDB: 5FW3)⁵⁰. Homology models were visualized using ChimeraX⁹⁷.