170128 20260318155255.0 doi 10.1038/s42004-026-01908-9 sideral 148656 ART-2026-148656 eng Doppler, Diandra Minimized sample consumption for time-resolved serial crystallography applied to the redox cycle of human NQO1 2026 Sample consumption for serial femtosecond crystallography with X-ray free electron lasers remains a major limitation preventing broader use in macromolecular crystallography. This drawback is exacerbated in time-resolved (TR) experiments, where the amount of sample required per reaction time point is multiplied by the number of time points investigated. To reduce this limitation, we demonstrate a segmented droplet generation strategy coupled to a mix-and-inject approach for TR studies at the European XFEL. The injector produces synchronized droplet trains that enable stable and reproducible injection of protein crystal slurries at significantly reduced flow rates. Using the human flavoenzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) as a test system, we collected diffraction data after mixing with NADH at 0.3 s and 1.2 s delays. The segmented injection approach achieved up to 97% reduction in sample consumption compared with continuous-flow injection while maintaining data quality suitable for TR crystallography. Reproducible electron density features consistent with low-occupancy NADH binding illustrate both the feasibility and the current limits of studying dynamic redox enzymes using this approach. This work establishes segmented droplet generation as a sample-efficient and XFEL-compatible method for future time-resolved serial crystallography experiments. Access copy available to the general public Unrestricted info:eu-repo/grantAgreement/ES/DGA/E35-23R info:eu-repo/grantAgreement/ES/MICINN/CNS2022-135713 info:eu-repo/grantAgreement/ES/MICINN/PID2022-136369NB-I00 info:eu-repo/grantAgreement/ES/MICINN/PID2023-151100NB-I00 info:eu-repo/semantics/openAccess by-nc-nd https://creativecommons.org/licenses/by-nc-nd/4.0/deed.es info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion Grieco, Alice Koh, Domin Manna, Abhik Ansari, Adil Alvarez, Roberto Karpos, Konstantinos Le, Hung Sonker, Mukul Ketawala, Gihan K. Mahmud, Samira Quereda-Moraleda, Isabel Sen, Sayantee Pey, Angel L. Letrun, Romain Dörner, Katerina Koliyadu, Jayanath C. P. de Wijn, Raphael Bielecki, Johan Han, Huijong Kim, Chan Koua, Faisal H. M. Round, Adam Sarma, Abhisakh Sato, Tokushi Schmidt, Christina Vakili, Mohammad Zabelskii, Dmitrii Bean, Richard Mancuso, Adrian P. Schulz, Joachim Fromme, Raimund Medina, Milagros Universidad de Zaragoza (orcid)0000-0001-8743-0182 Grant, Thomas D. Fromme, Petra Kirian, Richard A. Botha, Sabine Manuel Martin-Garcia, Jose Ros, Alexandra 1002 060 Universidad de Zaragoza Dpto. Bioq.Biolog.Mol. Celular Área Bioquímica y Biolog.Mole. 9, 1 (2026), 14 pp. Communications Chemistry 2399-3669 3341953 http://zaguan.unizar.es/record/170128/files/texto_completo.pdf Versión publicada 2774976 http://zaguan.unizar.es/record/170128/files/texto_completo.jpg?subformat=icon icon Versión publicada oai:zaguan.unizar.es:170128 articulos driver 2026-03-18-13:52:53 ARTICLE