Abstract As genetic code expansion advances beyond L-α-amino acids to backbone modifications and new polymerization chemistries, the field faces an increasingly broad challenge to discover what the ribosome can accommodate. Although the E. coli ribosome tolerates non-L-α-amino acids in vitro , few structural insights are available, and the boundary conditions for efficient bond formation are unknown. We describe a 2.1 Å cryo-EM structure of the E. coli ribosome containing well-resolved α-amino acid monomers coupled with a computational approach for which energy surface minima produced by metadynamics trend in agreement with established incorporation efficiencies. Reactive monomers across diverse structural classes favor a conformational space characterized by an A-site nucleophile to P-site carbonyl distance of &lt; 4 Å and a Bürgi-Dunitz angle of 90-110°. Monomers whose free energy minima fall outside these regions do not react. Application of this model should accelerate the in vivo and in vitro ribosomal synthesis and application of sequence-defined, non-peptide heterooligomers.

Atomistic simulations of the E. coli ribosome provide selection criteria for translationally active substrates