9EZR | pdb_00009ezr

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

Doppler, D., Grieco, A., Koh, D., Manna, A., Ansari, A., Alvarez, R., Karpos, K., Le, H., Sonker, M., Ketawala, G.K., Mahmud, S., Quereda-Moraleda, I., Sen, S., Pey, A.L., Letrun, R., Dorner, K., Koliyadu, J.C.P., de Wijn, R., Bielecki, J., Han, H., Kim, C., Koua, F.H.M., Round, A., Sarma, A., Sato, T., Schmidt, C., Vakili, M., Zabelskii, D., Bean, R., Mancuso, A.P., Schulz, J., Fromme, R., Medina, M., Grant, T.D., Fromme, P., Kirian, R.A., Botha, S., Manuel Martin-Garcia, J., Ros, A.

(2026) Commun Chem 

• PubMed: 41611948 Search on PubMed
• DOI: https://doi.org/10.1038/s42004-026-01908-9
• Primary Citation of Related Structures:  
  9EZQ, 9EZR, 9EZS, 9EZT, 9ID0


• PubMed Abstract: 
  

  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.

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Organizational Affiliation: 
• School of Molecular Sciences, Arizona State University, Tempe, AZ, USA.
Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, AZ, USA.
Department of Crystallography and Structural Biology, Institute of Physical Chemistry Blas Cabrera, Spanish National Research Council (CSIC), Madrid, Spain.
Department of Physics, Arizona State University, Tempe, AZ, USA.
Departamento de Química Física, Unidad de Excelencia en Química Aplicada a Biomedicina y Medioambiente e Instituto de Biotecnología, Universidad de Granada, Granada, Spain.
European XFEL GmbH, Schenefeld, Germany.
Center for Free-Electron Laser Science CFEL, Deutsches Elektronen-Synchrotron DESY, Hamburg, Germany.
Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, Australia.
Department of Biochemistry and Molecular and Cellular Biology, Faculty of Sciences and, Institute for Biocomputation and Physics of Complex Systems (BIFI), University of Zaragoza, Zaragoza, Spain.
Diamond Light Source Ltd, Harwell Science and Innovation Campus, Didcot, UK.
Department of Structural Biology, Jacobs School of Medicine and Biomedical Sciences, SUNY University at Buffalo, Buffalo, NY, USA.
Department of Crystallography and Structural Biology, Institute of Physical Chemistry Blas Cabrera, Spanish National Research Council (CSIC), Madrid, Spain. jmmartin@iqf.csic.es.
School of Molecular Sciences, Arizona State University, Tempe, AZ, USA. aros2@asu.edu.
Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, AZ, USA. aros2@asu.edu.
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