The emergence of antimicrobial resistance poses a major challenge to global health, yet the molecular mechanisms underlying resistance often remain incompletely understood. In this webinar, I will present a case study from my PhD research investigating the genetic and structural basis of gentamicin resistance in experimentally evolved bacterial mutants.

The project involved generating gentamicin-resistant mutants in the laboratory, performing whole-genome sequencing to identify resistance-associated mutations, and characterising the resulting protein variants. While mutations were identified across several proteins, the primary focus was on elongation factor G (EF-G), a key component of the bacterial translation machinery.

To investigate the structural consequences of these mutations, AlphaFold2 was used to predict mutant protein structures and compare them with their wild-type counterparts. Structural alignments revealed that some mutations produced subtle conformational shifts in domain IV of EF-G, an extended region located near the gentamicin-binding site on the ribosome, while other mutations showed little or no detectable structural change.

To place these findings into a functional context, experimental ribosome structures from the Protein Data Bank were integrated with AlphaFold predictions. By superimposing structures of EF-G-bound ribosomes and gentamicin-bound ribosomes, a composite structural model was generated, enabling the spatial relationship between resistance-associated mutations and the antibiotic-binding site to be examined. These analyses provided mechanistic insights into how specific mutations may reduce gentamicin binding through local structural and physicochemical effects.

The webinar will discuss the opportunities and limitations of using AlphaFold to investigate the structural consequences of genomic variation, the challenges of interpreting subtle structural differences, and the importance of integrating AI-predicted models with experimental structural data to generate biologically meaningful hypotheses.

This event is part of a broader webinar series on user cases of the AlphaFold resources. For more information about the series and its webinars, please visit the following link: AlphaFold in practice: research use cases | EMBL-EBI Training