Slow cooling of protein crystals.
Slow cooling of protein crystals.
复制标题
蛋白质晶体缓慢冷却。
DOI：
10.1107/s0021889809023553
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发表时间：
2009
影响因子：
6.1
通讯作者：
Thorne,RobertE
中科院分区：
文献类型：
--
作者：
Warkentin,Matthew;Thorne,RobertE
无冷冻保护剂的thumatin晶体以0.1 K s−1的速度从300 K冷却到100 K，比传统的闪速冷却慢103-104倍，同时连续收集x射线衍射数据，以便跟踪冷却过程中蛋白质晶格和溶剂性质的演变。衍射图显示在任何温度下都没有结晶冰的证据。这表明蛋白质分子的晶格本身就是一种优秀的冷冻保护剂，并且从1.5 M母液中加入酒石酸钠钾的冰成核率至少与70%甘油溶液中的冰成核率一样低。在缓慢冷却过程中，晶体质量仍然很高，在100 K（0.2°）时具有平均嵌合性。大部分嵌合性增加发生在~ 200 K以上，此时溶剂仍为液体，并且与单元胞的各向异性收缩同时发生。在180k附近发生了向类固体溶剂行为的交叉，并且在进一步冷却时没有进一步的晶体顺序退化。B因子随温度的变化表明蛋白质在210 K附近发生了动态转变，并且在较低温度下，斜率dB/dT比其他任何蛋白质都小3-6倍。这些结果确立了在300 ~ 100 K范围内进行蛋白质结构和动力学的完全温度控制研究的可行性。
Cryoprotectant-free thaumatin crystals have been cooled from 300 to 100 K at a rate of 0.1 K s−1 – 103–104 times slower than in conventional flash cooling – while continuously collecting X-ray diffraction data, so as to follow the evolution of protein lattice and solvent properties during cooling. Diffraction patterns show no evidence of crystalline ice at any temperature. This indicates that the lattice of protein molecules is itself an excellent cryoprotectant, and with sodium potassium tartrate incorporated from the 1.5 M mother liquor ice nucleation rates are at least as low as in a 70% glycerol solution. Crystal quality during slow cooling remains high, with an average mosaicity at 100 K of 0.2°. Most of the mosaicity increase occurs above ∼200 K, where the solvent is still liquid, and is concurrent with an anisotropic contraction of the unit cell. Near 180 K a crossover to solid-like solvent behavior occurs, and on further cooling there is no additional degradation of crystal order. The variation of B factor with temperature shows clear evidence of a protein dynamical transition near 210 K, and at lower temperatures the slope dB/dT is a factor of 3–6 smaller than has been reported for any other protein. These results establish the feasibility of fully temperature controlled studies of protein structure and dynamics between 300 and 100 K.