Weik M, Schreurs A M M, Leiros H K S, Zaccai G, Ravelli R B G, Gros P
Institut de Biologie Structurale, Laboratoire de Biophysique Moléculaire, Grenoble, France.
J Synchrotron Radiat. 2005 May;12(Pt 3):310-7. doi: 10.1107/S0909049505003316. Epub 2005 Apr 14.
The study of temperature-dependent physical changes in flash-cooled macromolecular crystals is pertinent to cryocrystallography and related issues such as crystal annealing, X-ray radiation damage and kinetic crystallography. In this context, the unit-cell volume of flash-cooled trigonal and orthorhombic trypsin crystals has been monitored upon warming from 100 to 200 K and subsequent re-cooling to 100 K. Crystals of both forms were obtained under the same crystallization conditions, yet they differ in solvent content and channel size. An abrupt non-reversible unit-cell volume decrease is observed at 185 K in orthorhombic and at 195 K in trigonal crystals as the temperature is increased; this result is consistent with ultra-viscous solvent leaving the crystals. Concomitant appearance of ice rings in the diffraction patterns suggests that the transported solvent forms crystalline ice. These results demonstrate that solvent in flash-cooled protein crystals is liquid-like near its crystallization temperature, as has been proposed, yet controversially discussed, for the case of pure water. The use of mineral oil prevents the unit-cell volume decrease in trigonal but not in orthorhombic crystals. The observation of liquid-like solvent has implications in the development of annealing protocols and points a way to the rational design of temperature-controlled crystallographic studies that aim either at studying specific radiation damage or at trapping enzymatic intermediate states.
对快速冷却的大分子晶体中温度依赖性物理变化的研究与低温晶体学以及诸如晶体退火、X射线辐射损伤和动力学晶体学等相关问题密切相关。在此背景下，已对快速冷却的三角晶系和正交晶系胰蛋白酶晶体从100 K升温至200 K并随后再冷却至100 K过程中的晶胞体积进行了监测。两种晶型的晶体是在相同的结晶条件下获得的，但它们在溶剂含量和通道尺寸方面存在差异。随着温度升高，在正交晶系晶体中于185 K时观察到晶胞体积突然出现不可逆减小，在三角晶系晶体中于195 K时出现这种情况；这一结果与超粘性溶剂离开晶体的情况一致。衍射图谱中同时出现的冰环表明所传输的溶剂形成了结晶冰。这些结果表明，快速冷却的蛋白质晶体中的溶剂在其结晶温度附近呈液态，正如针对纯水情况所提出的那样，但存在争议。使用矿物油可防止三角晶系晶体的晶胞体积减小，但对正交晶系晶体无效。液态溶剂的观察结果对退火方案的制定具有启示意义，并为合理设计旨在研究特定辐射损伤或捕获酶促中间态的温控晶体学研究指明了方向。
