De Carlo Sacha, Boisset Nicolas, Hoenger Andreas
Molecular, Cellular and Developmental Biology Department, Campus Box 347, University of Colorado at Boulder, Boulder, CO 80309-0347, USA.
Micron. 2008 Oct;39(7):934-43. doi: 10.1016/j.micron.2007.11.003. Epub 2007 Nov 17.
Cryo-negative staining was developed as a complementary technique to conventional cryo-electron microscopy on supramolecular complexes. It allows imaging biological samples in a comparable state of structural preservation to conventional cryo-EM but the staining produces better contrast in accessible areas and allows data recording at lower defocus values. Cryo-negative staining vitrifies biological particles in the presence of a concentrated ammonium molybdate solution at neutral pH. It was successfully used to study the structure and dynamics of several macromolecules, such as human transcription factors and RNA polymerases. Imaging macromolecular complexes with cryo-negative staining has been established previously to better than 2 nm detail. However, it has not been verified so far whether cryo-negative staining also visualizes secondary structure elements. Using the well known E. coli GroEL chaperonin, we could show that the structure is well preserved to approximately 10 A resolution. Secondary structure details are at least partially resolved in the electron density map.
冷冻负染色技术是作为超分子复合物常规冷冻电子显微镜的一种补充技术而开发的。它能使生物样品在结构保存状态上与传统冷冻电镜相当，但染色在可及区域产生更好的对比度，并允许在较低的散焦值下进行数据记录。冷冻负染色在中性pH值的浓钼酸铵溶液存在下使生物颗粒玻璃化。它已成功用于研究几种大分子的结构和动力学，如人类转录因子和RNA聚合酶。此前已利用冷冻负染色对大分子复合物进行成像，分辨率优于2纳米。然而，到目前为止，尚未证实冷冻负染色是否也能显示二级结构元件。利用著名的大肠杆菌GroEL伴侣蛋白，我们可以证明其结构在约10埃分辨率下保存良好。二级结构细节在电子密度图中至少部分得到解析。
