CryoEM on dark-state OaPAC

OaPAC is a light-activated cyanobacterial adenylate cyclase, which increases its cAMP production 20-fold upon illumination. It is a promising optogenetic tool. Time-resolved X- ray solution scattering reveals major light-induced conformational changes that are not visible in crystallography, likely due to crystal packing constraints. Preliminary cryo-EM data confirm that the dark-state structure resembles the crystallographic one, while illuminated and Q48E mutant samples show high flexibility, complicating 3D reconstruction. We aim to collect high-resolution cryo-EM datasets for dark and Q48E OaPAC. The Q48E mutant is functionally “trapped” in the light state, offering a unique chance to capture the active conformation. We will integrate molecular dynamics simulations results to help in the challenging single-particle analysis. This work will enable future time-resolved cryo-EM studies of OaPAC, advancing our understanding of light-driven enzymatic regulation.