Sample delivery methods for protein X-ray crystallography with a special focus on sample consumption

Abhik Manna, Diandra Doppler, Manasa P. Sripati, Mukul Sonker and Alexandra Ros ()
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Abhik Manna: Arizona State University
Diandra Doppler: Arizona State University
Manasa P. Sripati: Arizona State University
Mukul Sonker: Arizona State University
Alexandra Ros: Arizona State University

Nature Communications, 2025, vol. 16, issue 1, 1-29

Abstract: Abstract Serial crystallography (SX) has revolutionized structural biology by enabling high-resolution structure determination for important classes of proteins, including the study of relevant biomolecular reaction mechanisms. However, one of the ongoing challenges in this field remains the efficient use of precious macromolecule samples whose availability is often limited. Reducing sample consumption is thus critical in maximizing the potential of SX conducted at powerful X-ray sources such as synchrotrons and X-ray free-electron lasers (XFEL) to expand to a broader range of significant biological samples, gaining insights into unraveled biological reaction mechanisms. This review focuses on three primary sample delivery systems: fixed-targets, liquid injection, and hybrid methods, each with distinct advantages and limitations concerning sample consumption. The progress and challenges associated with these methods, highlighting advancements in reducing sample consumption and thus enabling the study of more diverse biological samples, are summarized. We compare the currently reported sample delivery methods in view of the minimum amount of sample required to obtain a full data set and discuss how the current approaches compare to this theoretical minimum. With this overview, we aim to provide a critical and comprehensive assessment of the current methods and experimental realizations for sample delivery in SX with proteins.

Date: 2025
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DOI: 10.1038/s41467-025-65173-5

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