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首页> 外文期刊>IUCrJ >Ice formation and solvent nanoconfinement in protein crystals
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Ice formation and solvent nanoconfinement in protein crystals

机译：蛋白质晶体中的冰形成和溶剂纳米约束

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Ice formation within protein crystals is a major obstacle to the cryocrystallographic study of protein structure, and has limited studies of how the structural ensemble of a protein evolves with temperature in the biophysically interesting range from ∼260 K to the protein–solvent glass transition near 200 K. Using protein crystals with solvent cavities as large as ∼70 Å, time-resolved X-ray diffraction was used to study the response of protein and internal solvent during rapid cooling. Solvent nanoconfinement suppresses freezing temperatures and ice-nucleation rates so that ice-free, low-mosaicity diffraction data can be reliably collected down to 200 K without the use of cryoprotectants. Hexagonal ice (Ih) forms in external solvent, but internal crystal solvent forms stacking-disordered ice (Isd) with a near-random stacking of cubic and hexagonal planes. Analysis of powder diffraction from internal ice and single-crystal diffraction from the host protein structure shows that the maximum crystallizable solvent fraction decreases with decreasing crystal solvent-cavity size, and that an ∼6 Å thick layer of solvent adjacent to the protein surface cannot crystallize. These results establish protein crystals as excellent model systems for the study of nanoconfined solvent. By combining fast cooling, intense X-ray beams and fast X-ray detectors, complete structural data sets for high-value targets, including membrane proteins and large complexes, may be collected at ∼220–240 K that have much lower mosaicities and comparable B factors, and that may allow more confident identification of ligand binding than in current cryocrystallographic practice.
机译：蛋白质晶体中的冰形成是蛋白质晶体低温晶体学研究的主要障碍，并且对蛋白质的结构整体如何随温度变化的生物物理研究有限，从大约260 K到大约200的蛋白质-溶剂玻璃转变温度K.使用溶剂腔大至70？Å的蛋白质晶体，使用时间分辨X射线衍射研究快速冷却过程中蛋白质和内部溶剂的响应。溶剂纳米约束抑制了冻结温度和冰成核速率，因此无需使用冷冻保护剂就可以可靠地收集低至200 K的无冰，低水分的衍射数据。六方冰（Ih）在外部溶剂中形成，但内部晶体溶剂形成堆积无序的冰（Isd），立方和六边形平面几乎随机堆积。从内部冰中进行的粉末衍射分析和从宿主蛋白质结构进行的单晶衍射分析表明，随着结晶溶剂腔尺寸的减小，最大可结晶溶剂分数降低，并且靠近蛋白质表面的〜6thickÅ厚的溶剂层无法结晶。这些结果将蛋白质晶体确立为研究纳米受限溶剂的优秀模型系统。通过结合快速冷却，强力X射线束和快速X射线检测器，可以在约220–240 K的温度下收集高价值目标的完整结构数据集，包括膜蛋白和大型复合物，这些镶嵌物的镶嵌率要低得多，并且可比B因素，与当前的低温晶体学实践相比，它可以更可靠地鉴定配体结合。

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