Structural Basis for C⁸ methylation of 23S ribosomal RNA by Cfr

Cfr methylates C⁸ of adenosine 2503 (A2503) in 23S ribosomal RNA (rRNA) and will also methylate C² of A2503 after methylating C⁸. C⁸ methylation confers resistance to more than five classes of clinically used antibiotics, highlighting it as a worrisome mechanism of antibiotic resistance. Here, we report the structure of Cfr, determined by cryogenic electron microscopy (Cryo-EM). Despite its small size (~36 kDa), we exploit a transient protein-RNA crosslink that forms during catalysis, which requires Cys105 to resolve. Using a Cfr Cys105Ala variant and an 87-nucleotide strand of rRNA, we isolate the crosslinked species and determine its structure to 3.0 Å resolution. Notably, the 87-mer rRNA adopts an L-shaped conformation characteristic of tRNAs, rather than the conformation it assumes in the ribosome.