TEMP DEPEND OF AVERAGE & SITE-SPECIFIC RADIATION DAMAGE IN PROTEIN CRYSTALS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们将尝试解决两个问题： 1.在T=100K(氮气)和T=20K(氦气)下采集的数据对蛋白质晶体的损伤与剂量有什么不同？有几个实验几乎或根本没有效果，但效果可能太小，无法用标准方法可靠地量化。博士后助理(现在是助理教授)Jan Kmetko开发了一种数据收集和分析方案，该方案应该能够准确可靠地解决分歧。在实验的这一部分，我们将在一个狭窄的角度楔形(5度)中对晶体进行剂量测定并收集数据，这将不足以进行完全的结构优化，剂量达到20毫灰或更多。 2.特定部位的辐射损害是否依赖于远低于水的玻璃化转变温度的温度？在目前研究蛋白质晶体辐射损伤的人中，有一种观点认为，在相对较低的剂量下观察到特定部位的损伤(例如，二硫键的断裂)是二次辐射损伤的证据。然而，我们认为(基于旧的ESR实验和基本考虑)这种损伤是通过电子转移发生的，而不是通过原子和分子自由基热扩散而发生的，因此是主要的损伤。即使特定位置的损伤完全是由于电子运动造成的，这种运动也可能通过隧穿或热跳跃发生。通过比较T=100K和T=20K对蛋白质晶体的定点损伤，我们希望能够确定定点损伤的性质。 这些实验将涉及从同一晶体上的同一地点获取一系列数据集(每个数据集都有足够的信息来评估二硫键的状态)，并观察随着总吸收剂量的增加，部位特定损伤的增长情况。将在T=100K和T=20K收集的数据进行比较，将会发现特定部位损坏的速度和程度有任何差异。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We will try to address two questions: 1. Is there any difference in damage versus dose to protein crystals when data is collected at T=100 K (nitrogen gas) and T=20 K (helium gas)? Several experiments have seen little or no effect, but the effects may be too small to be reliably quantified using standard methods. Postdoctoral Associate (now Assistant Professor) Jan Kmetko developed a data collection and analysis protocol that should give the accuracy to reliably resolve differences. In this part of the experiment, we will dose crystals and collect data in a narrow angular wedge (5 degrees), which will not be sufficient for a full structural refinement, out to doses of 20 MGray or more. 2. Does site-specific radiation damage depend on temperature at temperatures well below water's glass transition temperature? There is a belief among those currently studying radiation damage to protein crystals that the observation of site-specific damage (e.g., breakage of disulfide bonds) at relatively low doses is evidence for secondary radiation damage. However, we believe (based on old ESR experiments and basic considerations) that this damage occurs via electron transfer, not by thermal diffusion of atomic and molecular radicals, and therefore is primary damage. Even if site specific damage is due entirely to electron motion, this motion may occur via tunneling or thermal hopping. By comparing site-specific damage to protein crystals at T=100 K and T=20 K, we hope to be able to nail down the nature of site-specific damage. These experiments will involve taking a series of data sets (each with enough information to evalate the status of disulfide bonds) from the same spot on the same crystal , and watching the growth of site specific damage with total absorbed dose. Comparison of data collected at T=100 K and T=20 K will reveal any differences in the rate and extent of site specific damage.