Abstract
Transposition is a fundamental driver of genome evolution, enabling the movement of discrete DNA segments (transposons) within genomes. This process can modulate gene expression and contributes to the spread of antibiotic resistance and virulence factors. We recently determined different cryo-electron microscopy (cryo-EM) structures of the IS21 transposon, a widespread mobile genetic element that encodes IstA, a transposase, and IstB, a AAA + ATPase essential for DNA transposition. The reconstructions of these factors in the pre- and post-transposition states revealed key insights into the architecture and conformational dynamics of the transpososome. In parallel, we evaluated one of the newest structure prediction engines, AlphaFold3 (AF3), to help guide the structural work and explore its capacity to model these intricate protein-DNA assemblies. Here we present a focused comparison between the cryo-electron-microscopy structures of the IS21 transpososome and models generated with the public AF3 server. Overall, our findings show that while AF3 excels at predicting individual monomeric domains and select oligomeric arrangements, it struggles to capture some complex assemblies, conformational changes, and higher-order interactions critical for transposon function.