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的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 我们将试图解决两个问题： 1. 当在T = 100 K（氮气）和T = 20 K（氦气）下收集数据时，蛋白质晶体的损伤与剂量是否有任何差异？ 几个实验已经看到很少或没有影响，但影响可能太小，无法使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协议，该协议应该能够准确可靠地解决差异。 在实验的这一部分，我们将在一个狭窄的角度楔形（5度），这将不足以为一个完整的结构细化剂量的晶体和收集数据，到20 MGray或更多。 2.在远低于水的玻璃化转变温度的温度下，特定地点的辐射损伤是否取决于温度？ 目前研究蛋白质晶体辐射损伤的人认为，观察位点特异性损伤（例如，二硫键断裂）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的热扩散，因此是初级损伤。 即使位点特异性损伤完全是由于电子运动，这种运动也可能通过隧穿或热跳跃发生。 通过比较蛋白质晶体在T = 100 K和T = 20 K时的位点特异性损伤，我们希望能够确定位点特异性损伤的性质。 这些实验将涉及从同一晶体上的同一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），并观察总吸收剂量下特定部位损伤的增长。 在T = 100 K和T = 20 K时收集的数据的比较将揭示特定部位损伤的速率和程度的任何差异。

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.