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Flash cooling of macromolecular crystals was investigated by measurement of the cooling rates achieved by different cryoprotectant solutions (glycerol, sucrose and polyethylene glycol) with different sample volumes (0.2-0.8 mm3) and by different cooling agents (cold nitrogen or helium gas, liquid nitrogen and liquid propane). Samples were not cooled instantaneously; it usually took 0.5 to 1 s to reach the glass transition temperature and 1 to 2 s to reach the final temperature. The cooling rate varied from ~50 to 700 K s-1 depending on the volume of the sample, the cooling agent used and the temperature at the time of measurement. The cooling rate may affect the distribution of unit-cell dimensions, structural states and the average structure throughout the volume of the crystal, and hence its mosaic spread.
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