Flash-cooling of macromolecular crystals for X-ray diffraction analysis is usually performed in liquid nitrogen (LN2). Cryogens different than LN2 are used as well for this procedure but are highly underrepresented, e.g., liquid propane and liquid ethane. These two cryogens have significantly higher cooling rates compared with LN2 and may thus be beneficial for flash-cooling of macromolecular crystals. Flash-cooling in liquid propane or liquid ethane results in sample vitrification but is accompanied by solidification of these cryogens, which is not compatible with the robotic systems nowadays used for crystal mounting at most synchrotrons. Here we provide a detailed description of a new double-chambered device and procedure to flash-cool loop mounted macromolecular crystals in different cryogenic liquids. The usage of this device may result in specimens of better crystal- and optical quality in terms of mosaic spread and ice contamination. Furthermore, applying the described procedure with the new double-chambered device provides the possibility to screen for the best flash-cooling cryogen for macromolecular crystals on a routine basis, and, most importantly, the samples obtained allow the usage of state-of-the-art robotic sample-loading systems at synchrotrons.
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