Best practices for cryo‐trapping time‐resolved crystallography with the Spitrobot crystal plunger

作者：Robert Bosman, Caitlin E. Hatton, Andreas Prester, Maria Spiliopoulou, Virginia Apostolopoulou, Friedjof Tellkamp, P. Mehrabi, Eike C. Schulz · 发表于：Protein Science · 年份：2026 · DOI：10.1002/pro.70741 · 研究领域：Enzyme Structure and Function、Biochemical and Molecular Research、Advanced Electron Microscopy Techniques and Applications

Capturing meta-stable conformations of enzymes and ligand complexes demands structural snapshots beyond static crystal structures. While time-resolved serial crystallography at room temperature offers a time-resolution down to the femto-second domain, it requires large amounts of micro crystals, specialized beamlines, and considerable experience. Moreover, as the majority of enzymes display turnover-times in the millisecond domain or slower, simpler methods can provide meaningful structural insight into enzyme catalysis. Vitrification of protein crystals can trap reaction intermediates by rapid cooling to <100 K and has traditionally been used to gain insight into long lived reaction intermediates such as product complexes. However, manual vitrification procedures are limited to long delay times of at least several seconds and heavily suffer from operator variability. A solution to this problem is provided by automatic crystal plunging devices, such as the Spitrobot, that plunge loop-mounted protein crystals into liquid nitrogen within millisecond time-scales. Versatile means of reaction initiation can be achieved either by micro dispensing a ligand droplet, or via optical excitation of light-sensitive proteins, or via the photoactivation of caged compounds. In addition to the conceptual simplicity, another benefit of cryo-trapping is that data can be collected at conventional synchrotron beamlines, exploiting their robust high-throughput capabilities. Thus, compared to room-...