Antibiotic resistance is a global health threat. The long-term goal of my research is to synthesize novel natural product-like antibiotics using engineered enzymes and to uncover new antibiotic resistance mechanisms. To realize these goals, we are determining the mechanism of biosynthetic enzymes that construct polyketide antibiotics and proteins that confer resistance against DNA intercalator antibiotics. Structural biology is at the core of our research; we extensively use cryo-EM and X-ray crystallography. We often study the same protein using both techniques, and this approach has led to discoveries that could not have been made using either method alone. For Lsd14, a modular polyketide synthase that produces the polyketide antibiotic lasalocid A, the two techniques revealed Lsd14’s architecture at different steps of the catalytic cycle. For Ecm16, which provides resistance against the nonribosomal peptide antibiotic echinomycin, we obtained a high-resolution structure of the substrate-free Ecm16 using X-ray crystallography and a medium-resolution structure of the Ecm16-DNA-echinomycin complex using cryo-EM. Molecular knowledge gained from these studies is expected to produce new drugs and therapeutic strategies for combating antibiotic resistance.