Injector-based pump-probe serial synchrotron crystallography of CrPhotLOV1 at the MicroMAX beamline

Olasz, B.; Wisniewska, M.; Koczurowska, A.; Casadei, C. M.; Solecka, A.; Kapetanaki, S. M.; Pachota, M.; Lopes Cavalcante, L.; Asghar, A.; Lang, D.; Milas, M.; Ursby, T.; Shankar, M. K.; Chenchiliyan, M.; Nan, J.; Nogly, P.

doi:10.64898/2026.10.26.762041

bioRxiv · 10.64898/2026.10.26.762041

Injector-based pump-probe serial synchrotron crystallography of CrPhotLOV1 at the MicroMAX beamline

Olasz, B.·Wisniewska, M.·Koczurowska, A.·Casadei, C. M.·Solecka, A.·Kapetanaki, S. M.·Pachota, M.·Lopes Cavalcante, L.·Asghar, A.·Lang, D.·Milas, M.·Ursby, T.·Shankar, M. K.·Chenchiliyan, M.·Nan, J.·Nogly, P.

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Abstract

Time-resolved serial synchrotron crystallography (TR-SSX) has become a powerful approach for investigating structural dynamics in proteins under near-physiological conditions. Its successful application, however, depends on robust sample delivery, reliable pump-probe synchronization, and reproducible data-processing strategies. Here, an injector-based pump-probe TR-SSX workflow at the MicroMAX beamline is applied to investigate the structural dynamics of the LOV1 domain of the blue-light photoreceptor phototropin from Chlamydomonas reinhardtii (CrPhotLOV1). Room-temperature serial datasets were collected for an independently measured laser-off dark state together with four light-activated pump-probe time windows (0-350, 500-850, 1000-1350 and 1500-1850 {micro}s after a microsecond laser pulse). All four time windows and an interleaved pump-probe dark reference were recorded in a single run with the JUNGFRAU detector at 2 kHz. Because thioether adduct formation in CrPhotLOV1 is complete within about 20 {micro}s and the adduct persists for hundreds of seconds, the four delays probe the same photoadduct state and serve as independent replicates. The workflow was evaluated in terms of data quality, light contamination in the pump-probe dark reference, difference Fourier analysis, extrapolated electron-density maps and refinement of light-state models. Consistent activation levels were obtained across all time points, enabling reliable structural interpretation of the light-activated state. Difference Fourier maps and refined extrapolated models reproducibly revealed formation of the characteristic FMN-Cys57 thioether adduct together with localized rearrangements within the chromophore-binding pocket. The refined light-state structures agree closely with previously reported CrPhotLOV1 models. These results establish a robust and reproducible workflow for injector-based pump-probe TR-SSX at the MicroMAX beamline, suitable for routine time-resolved studies of light-responsive proteins.

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Olasz, B., Wisniewska, M., Koczurowska, A., Casadei, C. M., Solecka, A., Kapetanaki, S. M., Pachota, M., Lopes Cavalcante, L., Asghar, A., Lang, D., Milas, M., Ursby, T., Shankar, M. K., Chenchiliyan, M., Nan, J., Nogly, P.. 2026-10-30. Injector-based pump-probe serial synchrotron crystallography of CrPhotLOV1 at the MicroMAX beamline. https://doi.org/10.64898/2026.10.26.762041

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