Modular　synthesis　and　structural　biology　are　used　to　design　and　characterize　group　A　streptogramin　antibiotics,　one　of　which　has　activity　against　streptogramin-resistant　strains　and　demonstrates　efficacy　in　a　mouse　model　of　bacterial　infection.　Natural　products　serve　as　chemical　blueprints　for　most　antibiotics　in　clinical　use.　The　evolutionary　process　by　which　these　molecules　arise　is　inherently　accompanied　by　the　co-evolution　of　resistance　mechanisms　that　shorten　the　clinical　lifetime　of　any　given　class　of　antibiotics(1).　Virginiamycin　acetyltransferase　(Vat)　enzymes　are　resistance　proteins　that　provide　protection　against　streptogramins(2),　potent　antibiotics　against　Gram-positive　bacteria　that　inhibit　the　bacterial　ribosome(3).　Owing　to　the　challenge　of　selectively　modifying　the　chemically　complex,　23-membered　macrocyclic　scaffold　of　group　A　streptogramins,　analogues　that　overcome　the　resistance　conferred　by　Vat　enzymes　have　not　been　previously　developed(2).　Here　we　report　the　design,　synthesis,　and　antibacterial　evaluation　of　group　A　streptogramin　antibiotics　with　extensive　structural　variability.　Using　cryo-electron　microscopy　and　forcefield-based　refinement,　we　characterize　the　binding　of　eight　analogues　to　the　bacterial　ribosome　at　high　resolution,　revealing　binding　interactions　that　extend　into　the　peptidyl　tRNA-binding　site　and　towards　synergistic　binders　that　occupy　the　nascent　peptide　exit　tunnel.　One　of　these　analogues　has　excellent　activity　against　several　streptogramin-resistant　strains　ofStaphylococcus　aureus,　exhibits　decreased　rates　of　acetylation　in　vitro,　and　is　effective　at　lowering　bacterial　load　in　a　mouse　model　of　infection.　Our　results　demonstrate　that　the　combination　of　rational　design　and　modular　chemical　synthesis　can　revitalize　classes　of　antibiotics　that　are　limited　by　naturally　arising　resistance　mechanisms.