Hu Minghui, Qian Luping, Briñas Raymond P, Lymar Elena S, Kuznetsova Larisa, Hainfeld James F
Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA.
J Struct Biol. 2008 Jan;161(1):83-91. doi: 10.1016/j.jsb.2007.09.023. Epub 2007 Oct 11.
Cryo-electron microscopy single particle analysis shows limited resolution due to poor alignment precision of noisy images taken under low electron exposure. Certain advantages can be obtained by assembling proteins into two-dimensional (2D) arrays since protein particles are locked into repetitive orientation, thus improving alignment precision. We present a labeling method to prepare protein 2D arrays using gold nanoparticles (NPs) interconnecting genetic tag sites on proteins. As an example, mycobacterium tuberculosis 20S proteasomes tagged with 6x-histidine were assembled into 2D arrays using 3.9-nm Au NPs functionalized with nickel-nitrilotriacetic acid. The averaged top-view images from the array particles showed higher resolution (by 6-8A) compared to analysis of single particles. The correct 7-fold symmetry was also evident by using array particles whereas it was not clear by analysis of a comparable number of single particles. The applicability of this labeling method for three-dimensional reconstruction of biological macromolecules is discussed.
冷冻电子显微镜单颗粒分析显示，由于在低电子曝光下拍摄的噪声图像对齐精度较差，分辨率有限。将蛋白质组装成二维（2D）阵列可获得某些优势，因为蛋白质颗粒被锁定在重复的方向上，从而提高了对齐精度。我们提出了一种标记方法，使用连接蛋白质上基因标签位点的金纳米颗粒（NPs）制备蛋白质二维阵列。例如，用6x-组氨酸标记的结核分枝杆菌20S蛋白酶体，使用用次氮基三乙酸镍功能化的3.9纳米金纳米颗粒组装成二维阵列。与单颗粒分析相比，阵列颗粒的平均顶视图图像显示出更高的分辨率（提高了6-8埃）。使用阵列颗粒时，正确的7重对称性也很明显，而分析相当数量的单颗粒时则不明显。讨论了这种标记方法在生物大分子三维重建中的适用性。
