Research Article: Density and electron density of aqueous cryoprotectant solutions at cryogenic temperatures for optimized cryoprotection and diffraction contrast

Date Published: May 01, 2018

Publisher: International Union of Crystallography

Author(s): Timothy J. Tyree, Ritwik Dan, Robert E. Thorne.


The densities of aqueous solutions of eight common cryoprotectants were measured at T = 77 K and were used to determine electron densities at T = 77 K and thermal contractions on cooling from room temperature. The results provide a quantitative basis for choosing cryoprotectants to optimize outcomes in cryocrystallography, cryo-SAXS, cryogenic temperature X-ray imaging and vitrification-based biological cryopreservation.

Partial Text

The formation and physical properties of crystalline and glassy/vitreous/amorphous phases of water and of aqueous solutions are important in several areas of science and technology. In biological cryopreservation, ice crystals that form within cells can puncture membranes and damage other cellular components. Ice-crystal growth concentrates solutes in the remaining liquid, sometimes driving protein aggregation and/or denaturation (Fahy & Wowk, 2015 ▸). In biomolecular X-ray crystallography, the formation of internal and external ice damages crystals, increasing their mosaicity and reducing resolution (Rupp, 2009 ▸). In cryogenic temperature small-angle X-ray scattering (cryo-SAXS), large scattering at small wavevectors q (i.e. at small scattering angles 2θ) by even minute amounts of ice can overwhelm scattering from the biomolecule of interest (Meisburger et al., 2013 ▸). Ice formation is also a critical problem in cryogenic temperature X-ray imaging of, for example, hydrated cells (Huang et al., 2009 ▸; Rodriguez et al., 2015 ▸; Lima et al., 2009 ▸), and in cryo-electron microscopy (cryo-EM; Costello, 2006 ▸), especially in high-resolution single-particle cryo-EM, where diffraction images of enormous numbers of molecules must be combined to generate high-resolution structures. Even when crystalline ice does not form, thermal contraction or expansion on cooling to the glass phase can damage samples (Juers & Matthews, 2004 ▸; Kriminski et al., 2002 ▸; Rabin et al., 2006 ▸; Hopkins et al., 2015 ▸). Differential contraction between internal and external solvent and protein crystals, between regions of a cell or tissue having different solvent contents, between cryo-SAXS samples and their holders, and between thin-film X-ray imaging and cryo-EM samples and their supports can cause sample deformation, creep, fracturing and microscale disorder. Solvent contraction or expansion on cooling also modulates the solvent electron density, and this may have large effects on the strength of the diffraction signal from biomolecules in cryo-SAXS and cryo-EM.

Cryoprotectant solutions were prepared as described in the Supporting Information, giving typical concentration uncertainties of ∼1%(w/w) for methanol, ethanol and 2-propanol and <0.1%(w/w) for the less volatile CPAs. The present results provide quantitative data and fits for optimizing cryoprotectant choice and concentration in cryocrystallography, cryo-SAXS, cryogenic temperature X-ray imaging and vitrification-based protocols for single-cell cryopreservation, given constraints on cooling rates, sample thermal contraction and/or electron-density contrast between biomolecules and the solvent.   Source:


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