Abstract
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.
Highlights
Structure solution of macromolecules by X-ray crystallography requires the production of 3-dimensional (3D) crystals, which are nowadays almost exclusively analyzed using intense Xray synchrotron radiation
In order to assess the influence of the latter on crystal quality, ice contamination of the specimen and the achieved resolution, we here describe an adapted macromolecular flash-cooling procedure based on a newly designed double-chambered device. This device renders the flash-cooling of macromolecular crystals in liquid propane or liquid ethane to a method, which is efficient and compatible with state-ofthe-art robotic crystal mounting systems used at synchrotrons
We designed a double-chambered device (Fig 1), which can be used on a routine basis to flashcool macromolecular crystals using different cryogenic liquids
Summary
Structure solution of macromolecules by X-ray crystallography requires the production of 3-dimensional (3D) crystals, which are nowadays almost exclusively analyzed using intense Xray synchrotron radiation. To reduce radiation damage of the specimen during diffraction experiments, analyses are carried out at cryogenic temperatures, which leads to datasets of better quality [1]. To prevent ice formation, successful crystal flash-cooling protocols include the addition of cryoprotectants such as glycerol or polyethylene glycol (PEG) to the crystallization solution [2]. After successful crystal growth and cryo-condition screening [3], the specimen is flash-cooled in a cryogenic liquid, and either directly analyzed or stored at cryogenic temperatures until the diffraction experiment is conducted.
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