New Predictive Methods for Cryoprotection in Macromolecular Crystallography

描述（由申请人提供）：大分子结构的知识对于理解和开发各种疾病的治疗方法非常重要。大多数大分子结构都是用x射线晶体学来确定的，其中一个关键步骤是将晶体低温冷却到~100 K，以减轻电离辐射对晶体的损伤。为了成功冷却，通常使用主要是反复试验的方法对晶体进行预处理（冷冻保护），并且通常需要进行广泛的筛选。在这里，我们提出了一种新的方法，该方法基于对感兴趣的晶体的简单测量来预测最佳的冷冻保护方案。最好的冷冻保护溶液是从我们所描述的30多个列表中选择的，这样的冷冻溶液的热收缩正确地补偿了晶体固有的热收缩。在目标1中，我们对已知的蛋白质晶体进行了调查，挑选晶体在随后的目标中进行测试，这些目标跨越了一系列可能对冷冻结果产生影响的物理特性。目的2测试了预测晶体内部最佳冷冻溶液的特定方法。目的3，测试一种类似的方法来预测晶体外部的最佳冷冻溶液。最后，在Aim 4中，进行了域分析测量，以了解冷却诱导损伤的细节，因为冷冻溶液是优化的。在晶体学领域广泛采用有效的冷冻保护优化预测方法的长期影响将是确保从每个晶体中记录尽可能高质量的衍射。这将(a)提高x射线晶体学测定的结构的平均质量，(b)使更困难的晶体学问题更容易处理。这项工作将通过提高基于大分子结构的生物解释的可靠性来影响公共卫生，并将增加有效确定对健康有潜在影响的任何特定分子结构的可能性。从长远来看，这将提高我们对由遗传或环境因素引起的大分子结构和功能改变引起的疾病的病因的理解，并促进治疗。

DESCRIPTION (provided by applicant): Knowledge of macromolecular structure is important for understanding and developing treatments for a diverse array of diseases. Most macromolecular structures are determined with X-ray crystallography, for which a key step is to cryogenically cool the crystals to ~100 K in order to mitigate crystal damage from the ionizing radiation. For successful cooling, the crystals are usually pretreated (cryoprotected) using methods that are mainly trial and error and often require extensive screening. Here we propose a new approach in which the optimal cryoprotection scheme is predicted based on simple measurements on the crystal of interest. The best cryoprotective solution is chosen from a list of more than 30 we have characterized such that the thermal contraction of the cryosolution correctly compensates for the inherent thermal contraction of the crystal. In Aim 1 we perform a survey of known protein crystals, picking crystals to test in subsequent aims that span a range of physical characteristics likely to have an effect on the cryo-cooling result. Aim 2 tests a specific method for predicting the optimal cryosolution internal to the crystal. Aim 3, tests a similar method to predict the optimal cryosolution external to the crystal. Finally, in Aim 4 domain analysis measurements are performed to understand the details of the cooling- induced damage as the cryosolution is optimized. The long term effects of the widespread adoption of effective predictive methods of cryoprotection optimization on the field of crystallography would be to ensure that the highest quality diffraction possible is recorded from each crystal. This would (a) improve the average quality of structures determined by X-ray crystallography and (b) make the more difficult crystallographic problems more tractable. This work will impact public health by improving the reliability of biological interpretations based on macromolecular structures, and will increase the likelihood that the structure of any particular molecule with a potential impact on health can be efficiently determined. In the long term, this would improve our understanding of the causes of, and facilitate treatments for, diseases resulting from the alteration of macromolecular structure and function by either genetic or environmental factors. PUBLIC HEALTH RELEVANCE: High resolution three-dimensional structure determination of proteins, key for understanding the molecular basis of disease, are usually carried out at low temperatures. Cooling the samples is a delicate process with a high likelihood of failure. We have developed a method designed to make the cooling process more straightforward and predictable.