Mass Mapping of Macromolecular Assemblies

大分子组装体的质量作图

• 批准号: 6432957
• 负责人: Richard D Leapman
• 金额: --
• 依托单位: OFFICE OF THE DIRECTOR, NATIONAL INSTITUTES OF HEALTH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6432957
• 关键词: calcium; calmodulin dependent protein kinase; digital imaging; enzyme activity; macromolecule; molecular weight; phosphorylation; scanning transmission electron microscopy; spectrometry; structural biology

Scanning transmission electron microscopy (STEM) provides a versatile method for determining the molecular mass and hence the arrangement of subunits in large protein assemblies. Macromolecules are adsorbed onto a thin support film and a nanometer-sized electron probe is scanned across the specimen while the elastic scattering signal is collected. The resulting digital image intensity is proportional to the local mass density of the specimen. Images are recorded at low electron dose by counting single pulses from an annular detector positioned after the specimen. STEM can be applied directly to individual macromolecules in a heterogeneous population and is not therefore subject to averaging artifacts. Mass determination can often be made to an accuracy of 1 or 2% based on analysis of only 1000 molecules, which corresponds to about 1 femtogram of material, although in practice several nanograms are currently required to prepare a suitable specimen.

扫描透射电子显微镜（STEM）提供了一种多功能方法，用于确定分子质量，从而在大蛋白质组件中的亚基排列。大分子被吸附到薄的支撑膜上，并在收集弹性散射信号的同时扫描了纳米尺寸的电子探针。 所得的数字图像强度与试样的局部质量密度成正比。图像是通过计算样品后位置的环形检测器的单个脉冲来记录低电子剂量的。茎可直接应用于异质种群中的单个大分子，因此不受平均伪影的约束。 基于对1000个分子的分析，通常可以将质量测定的精度为1％或2％，这对应于大约1个材料图，尽管实际上目前需要几种纳米图来准备合适的样品。