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While time\u2010resolved serial crystallography at room temperature offers a time\u2010resolution down to the femto\u2010second domain, it requires large amounts of micro crystals, specialized beamlines, and considerable experience. Moreover, as the majority of enzymes display turnover\u2010times 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 &lt;100\u2009K 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\n                    <jats:italic>Spitrobot<\/jats:italic>\n                    , that plunge loop\u2010mounted protein crystals into liquid nitrogen within millisecond time\u2010scales. Versatile means of reaction initiation can be achieved either by micro dispensing a ligand droplet, or via optical excitation of light\u2010sensitive proteins, or via the photoactivation of caged compounds. In addition to the conceptual simplicity, another benefit of cryo\u2010trapping is that data can be collected at conventional synchrotron beamlines, exploiting their robust high\u2010throughput capabilities. Thus, compared to room\u2010temperature time\u2010resolved crystallography (RT\u2010TRX), users not only benefit from uncoupling sample\u2010preparation and data\u2010collection, but also from a reduction in the required technical expertise and ready access to radiation sources. However, as cryo\u2010trapping crystallography explores dynamic structural changes that become only visible by the comparison of several samples, experiments have to be carefully planned to carry out the necessary controls and to avoid mis\u2010 or over\u2010interpretation of the results. Here we describe a detailed protocol for cryo\u2010trapping time\u2010resolved crystallography using automated crystal\u2010plungers that enables researchers to map enzymatic reaction coordinate pathways within the millisecond domain.\n                  <\/jats:p>","DOI":"10.1002\/pro.70741","type":"journal-article","created":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T10:04:02Z","timestamp":1789466642000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Best practices for cryo\u2010trapping time\u2010resolved crystallography with the Spitrobot crystal plunger"],"prefix":"10.1002","volume":"35","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9939-602X","authenticated-orcid":false,"given":"Robert","family":"Bosman","sequence":"first","affiliation":[{"name":"Institute of Biochemistry and Signal Transduction University Medical Center Hamburg\u2010Eppendorf  Hamburg 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