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<p>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)&#xa0;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.</p><p></p>

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Minimized sample consumption for time-resolved serial crystallography applied to the redox cycle of human NQO1

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年1月29日

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  • Diandra Doppler,
  • Alice Grieco,
  • Domin Koh,
  • Abhik Manna,
  • Adil Ansari,
  • Roberto Alvarez,
  • Konstantinos Karpos,
  • Hung Le,
  • Mukul Sonker,
  • Gihan K. Ketawala,
  • Samira Mahmud,
  • Isabel Quereda-Moraleda,
  • Sayantee Sen,
  • Angel L. Pey,
  • Romain Letrun,
  • Katerina Dörner,
  • Jayanath C. P. Koliyadu,
  • Raphael de Wijn,
  • Johan Bielecki,
  • Huijong Han,
  • Chan Kim,
  • Faisal H. M. Koua,
  • Adam Round,
  • Abhisakh Sarma,
  • Tokushi Sato,
  • Christina Schmidt,
  • Mohammad Vakili,
  • Dmitrii Zabelskii,
  • Richard Bean,
  • Adrian P. Mancuso,
  • Joachim Schulz,
  • Raimund Fromme,
  • Milagros Medina,
  • Thomas D. Grant,
  • Petra Fromme,
  • Richard A. Kirian,
  • Sabine Botha,
  • Jose Manuel Martin-Garcia,
  • Alexandra Ros

摘要

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.