BACKGROUND Synchrotron radiation facilities are pillars of modern structural biology. Small-Angle X-ray scattering performed at synchrotron sources is often used to characterize the shape of biological macromolecules. A major challenge with high-energy X-ray beam on such macromolecules is the perturbation of sample due to radiation damage. RESULTS By employing atomic force microscopy, another common technique to determine the shape of biological macromolecules when deposited on flat substrates, we present a protocol to evaluate and characterize consequences of radiation damage. It requires the acquisition of images of irradiated samples at the single molecule level in a timely manner while using minimal amounts of protein. The protocol has been tested on two different molecular systems: a large globular tetremeric enzyme (β-Amylase) and a rod-shape plant virus (tobacco mosaic virus). Radiation damage on the globular enzyme leads to an apparent increase in molecular sizes whereas the effect on the long virus is a breakage into smaller pieces resulting in a decrease of the average long-axis radius. CONCLUSIONS These results show that radiation damage can appear in different forms and strongly support the need to check the effect of radiation damage at synchrotron sources using the presented protocol.

中文翻译：

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结合小角度X射线溶液散射和原子力显微镜来表征生物大分子上的辐射损伤。

背景技术同步辐射装置是现代结构生物学的支柱。在同步加速器源处进行的小角度X射线散射通常用于表征生物大分子的形状。在这种大分子上使用高能X射线束的主要挑战是由于辐射损伤而引起的样品扰动。结果通过采用原子力显微镜，另一种常用技术来确定沉积在平坦基底上的生物大分子的形状，我们提出了一种协议来评估和表征辐射损伤的后果。它要求在使用最小量蛋白质的情况下及时获取单分子水平的辐照样品的图像。该协议已在两种不同的分子系统上进行了测试：一种大的球状四聚体酶（β-淀粉酶）和一种杆状植物病毒（烟草花叶病毒）。球状酶的辐射损伤导致分子大小的明显增加，而对长病毒的影响是断裂成较小的碎片，导致平均长轴半径减小。结论这些结果表明，辐射损伤可以以不同的形式出现，并强烈支持使用提出的协议检查同步加速器源处辐射损伤的影响的需求。