Room-temperature light-state structure of the AsLOV2-Rac1 optogenetic actuator by sheet-on-sheet serial femtosecond crystallography

Room-temperature dark and light-state structures of the AsLOV2-Rac1 optogenetic fusion (PA-Rac1) were solved at 1.8 Å, revealing light-driven chromophore-environment and AsLOV2 loop changes.

• Why it matters: The activation mechanism of PA-Rac1 has been inferred mainly from cryogenic and isolated-domain studies, leaving uncertainty about how the fusion behaves at physiological temperatures.
• What they did: Serial femtosecond crystallography at SwissFEL Cristallina with sheet-on-sheet chip delivery used repeated 450 nm pumping to accumulate the FMN-cysteine adduct in ~80% of molecules and generate strong Fourier difference maps.
• The result: Light-state differences above 0.3 Å were confined to the chromophore environment, the D helix, and the Gβ-Hβ and Hβ-Iβ loops, including an Ile445 rotamer switch, while Rac1’s GTP/Mg2+ and switch regions showed no detectable light-induced change.

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