Last but not least, we purified individual human tRNAs directly from human cells and analyzed by single-particle cryo-EM to illustrate the potential of the approach as a future diagnostic tool.

We analyzed the local effects also in our cryo-EM maps and by using a newly developed biophysical method.

The team of @jmbujnicki.bsky.social used MD simulations to analyze the effect of specific Ψ-incorporation at positions 13 and 55 to understand the reasons behind the stabilizing effects on the tRNA molecules.

Our work is the first study to provide structures of several tRNAs before and after the introduction of modifications at specific sites. In detail, we analyzed 4 human tRNA species to decipher structural and stability effects induced by the installation of Ψ at single or multiple sites.

Foremost, our work also demonstrates the feasibility of tracking subtle conformational changes in tRNAs after getting modified. To achieve this, we purified all five human pseudouridine synthases (PUS) and generated tRNAs carrying different modification patterns.

We used single particle cryo-EM to directly visualize several human tRNAs and analyze the effects of individual tRNA modifications. To our knowledge, the work presents interpretable maps of the smallest asymmetric particles analyzed by single-particle cryo-EM so far.