MASS MEASUREMENT OF SINGLE MOLECULES WITH THE STEM

扫描透射电子显微镜(STEM)可以提供低剂量、 未染色生物样本的高分辨率图像。孤立对象 或者可以集成离散的图像特征(在减去 底物)来确定分子量，因此提供了直接联系 在生物化学和电子显微镜之间。STEM的精确度 粒细胞重量从10kD时的约20%提高到100kD时的约4% 对于在一年中成像的单个粒子，优于100万D以上的1% 剂量为10埃/埃的平方。TRNA、核小体、 纤维蛋白原、RNA聚合酶、谷氨酰胺合成酶、30S和50S核糖体 亚基、丝状病毒和烟草花叶病毒与 理论上的预测。平均值的标准误差可以减少 平均超过100-1000个粒子。粒子质量值的直方图可以 可用于表征制剂的均一性。这项技术 已常规应用于各种用户/协作者的 布鲁克海文STEM生物技术资源的过去几年的样本。 我们计划继续这些富有成效的合作和服务 活动，同时提炼技术的实用方面。进一步 将样品温度降低到20K有望减少质量损失 在120K时小于0.25%/el Angstroms的平方(比300K时小8倍 K)，允许更高的剂量和更高的准确度。曲面的缩减 较低温度下的迁移预计会导致较少的再分配 在辐射损伤期间的质量。这一预测将通过以下方式进行验证 已知结构的对照标本(如TMV)。 一个主要的新推动力将涉及使用图像平均程序来改善 低剂量图像的信噪比。这应该允许延长 已经在纤维蛋白原和动力蛋白上显示了域质量图谱 允许检测小分子(1-5kD)与络合物的结合 如谷氨酰胺合成酶和乙酰胆碱受体。对于粒子 对于球面或柱面对称，这些程序也将允许 作为半径函数的质量的测定。

The scanning transmission electron microscope (STEM) can provide low dose, high resolution images of unstained biological samples. Isolated objects or discrete image features can be integrated (after subtraction of substrate) to determine molecular weights and so provide a direct link between biochemistry and electron microscopy. The accuracy of STEM melecular weights improves from about 20% at 10 kD to about 4% at 100 kD and better than 1% above one million D for single particles imaged at a dose of 10 el/Angstroms squared. Practical results on tRNA, nucleosomes, fibrinogen, RNA polymerase, glutamine synthetase, 30s and 50s ribosomal subunits, filamentous viruses and TMV are in good agreement with theoretical predictions. Standard error of the mean can be reduced by averaging over 100-1,000 particles. Histograms of particle mass values can be used to characterize the homogeneity of the preparation. This technique has been applied routinely to a wide variety of users'/collaborators' samples over the past years at the Brookhaven STEM Biotechnology Resource. We propose to continue these productive collaborations and service activities while refining practical aspects of the technique. Further reduction of specimen temperature to 20 K is expected to reduce mass loss below 0.25%/el Angstroms squared now found at 120 K (8x less than at 300 K), permitting higher dose and higher accuracy. Reduction of surface migration at lower temperature is expected to lead to less redistribution of mass during radiation damage. This prediction will be tested using control specimens of known structure (e.g. TMV). A major new thrust will involve use of image averaging programs to improve signal to noise ratio in low dose images. This should permit extension of domainal mass mapping already demonstrated on fibrinogen and dynein to allow detection of the binding of small molecules (1-5 kD) to complexes such as glutamine synthetase and acetylcholine receptor. For particles with spherical or cylindrical symmetry, these programs will also permit determination of mass as a function of radius.