Methicillin resistant Staphylococcus aureus (MRSA) is a bacterial pathogen that presents genuine health concern. Treatment requires use of last line antibiotics such as members of the oxazolidone family, of which linezolid is the first member of this class to see regular use in the clinic. This work reports a small-scale linezolid resistance selection experiment whereby strains of MRSA were subjected to antibiotic selection. Clonal isolates that
exhibited linezolid resistance were isolated and their genomes were sequenced. Resistant mutants were isolated which accumulated mutations in the ribosomal protein uL3. A particular clone which had two mutations in uL3 exhibited resistance to linezolid, two-fold higher than the clinical breakpoint. Ribosomes from this strain were isolated and subjected to single particle cryo electron microscopic analysis and compared to the parent strain. It was found that the mutations in uL3 lead to a rearrangement of a loop that makes contact with Helix90, propagating a structural change over 15 Å away to the binding site of the antibiotic, which swings U2504 into the binding site of the antibiotic, leading to antibiotic resistance.