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

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. Sample consumption is one of the major bottlenecks limiting protein structure elucidation in serial crystallography. This review provides a critical and comprehensive assessment of state-of-the-art sample delivery approaches, discusses their realization and applications and delineates advancements in the field to reduce sample consumption.

• Publication year

  2025

• Venue

  Nature Communications

• Publication date

  2025-11-07

• Fields of study

  Biology, Medicine, Materials Science, Chemistry

• Identifiers
  DOI 10.1038/s41467-025-65173-5PMID 41203635PMCID 12594859
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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