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Flash-cooling of macromolecular crystals often compromises diffraction quality by increasing the mosaicity. In some cases, cycling the crystal between low temperature (LT) and room temperature (RT) can reverse this increase in mosaicity. Previous studies of RT/LT cycling have focused on the quality of the crystal as it was repeatedly returned to the LT state. Here, crystal quality is explored not only at LT but also when the crystal is returned to RT. The domain model is used to extract information about crystal order from reflection profiles measured from crystals of Escherichia coli β-galactosidase at both temperatures. Despite optimization of the cryocooling protocol, the mosaicity increases by about sixfold with cooling and is anisotropic at both temperatures. The mosaicity increase is the consequence of a decrease in domain volume, an increase in the variation of domain cell dimensions and an increase in the angular spread between domains. Upon rewarming, the mosaicity recovers substantially, including the somewhat surprising recovery of domain volume, but incompletely. Over multiple RT/LT cycles disorder in both states increases, which appears to mainly arise from radiation damage, although a contribution from cool–thaw processes cannot be ruled out. The analysis further suggests that LT disorder is governed by variability inherent in the cooling process combined with the overall history of the crystal. In contrast, RT disorder appears to be governed principally by the overall history of the crystal. This suggests that with these particular crystals under the experimental conditions used, particularly at high-intensity synchrotron X-ray sources, RT/LT cycling annealing protocols should involve few cycles so as to limit the hysteresis in both temperature states while taking advantage of the inherent variability in the cooling process that may result in improved crystal order at LT.

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