Experimental determination of the radiation dose limit for cryocooled protein crystals
Experimental determination of the radiation dose limit for cryocooled protein crystals
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DOI：
10.1073/pnas.0600973103
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发表时间：
2006-03-28
影响因子：
11.1
通讯作者：
Garman, EF
中科院分区：
文献类型：
--
作者：
Owen, RL;Rudiño-Piñera, E;Garman, EF
X 射线结构测定过程中低温冷却蛋白质晶体的辐射损伤已成为第三代同步加速器大分子衍射数据收集的固有部分。一般来说，辐射损伤是实验中不受欢迎的组成部分，可能会导致最终模型中出现错误的结构细节。因此，辐射损伤的表征已成为结构生物学家的一个重要领域。计算出的剂量限制为 2 x 10(7) Gy，使冷冻蛋白质晶体的衍射能力下降一半，已在第三代同步加速器源上进行了实验评估。从四个全铁蛋白和三个脱铁铁蛋白晶体中收集了连续的数据集。在测量入射光子通量并通过微粒诱导的 X 射线发射确定元素晶体成分后，使用程序 RADDOSE 计算每个晶体的吸收剂量。然后，可以将同步加速器辐射引起的衍射质量下降和特定结构变化直接与不同剂量/剂量率方案的吸收剂量进行比较：观察到剂量率增加 10 倍，寿命减少 10%。对于将衍射强度降低一半（D-1/2）所需的剂量，观察到相同类型的不同晶体之间以及脱铁铁蛋白和全铁蛋白之间的显着一致性。从这些测量中，获得了 D-1/2 = 4.3 (+/- 0.3) x 10(7) Gy 的剂量限制。然而，通过考虑其他数据质量指标，建议强度降低至 I-In2 = In2 x I-0，对应于 3.0 x 10(7) Gy 的吸收剂量，作为典型大分子晶体学实验的适当剂量限制。
Radiation damage to cryocooled protein crystals during x-ray structure determination has become an inherent part of macromolecular diffraction data collection at third-generation synchrotrons. Generally, radiation damage is an undesirable component of the experiment and can result in erroneous structural detail in the final model. The characterization of radiation damage thus has become an important area for structural biologists. The calculated dose limit of 2 x 10(7) Gy for the diffracting power of cryocooled protein crystals to drop by half has been experimentally evaluated at a third-generation synchrotron source. Successive data sets were collected from four holoferritin and three apoferritin crystals. The absorbed dose for each crystal was calculated by using the program RADDOSE after measurement of the incident photon flux and determination of the elemental crystal composition by micro-particle-induced x-ray emission. Degradation in diffraction quality and specific structural changes induced by synchrotron radiation then could be compared directly with absorbed dose for different dose/dose rate regimes: a 10% lifetime decrease for a 10-fold dose rate increase was observed. Remarkable agreement both between different crystals of the same type and between apoferritin and holoferritin was observed for the dose required to reduce the diffracted intensity by half (D-1/2). From these measurements, a dose limit of D-1/2 = 4.3 (+/- 0.3) x 10(7) Gy was obtained. However, by considering other data quality indicators, an intensity reduction to I-In2 = In2 x I-0, corresponding to an absorbed dose of 3.0 x 10(7) Gy, is recommended as an appropriate dose limit for typical macromolecular crystallography experiments.