Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany.
Elife. 2016 Mar 7;5:e13046. doi: 10.7554/eLife.13046.
We present a method for in-focus data acquisition with a phase plate that enables near-atomic resolution single particle reconstructions. Accurate focusing is the determining factor for obtaining high quality data. A double-area focusing strategy was implemented in order to achieve the required precision. With this approach we obtained a 3.2 Å resolution reconstruction of the Thermoplasma acidophilum 20S proteasome. The phase plate matches or slightly exceeds the performance of the conventional defocus approach. Spherical aberration becomes a limiting factor for achieving resolutions below 3 Å with in-focus phase plate images. The phase plate could enable single particle analysis of challenging samples in terms of small size, heterogeneity and flexibility that are difficult to solve by the conventional defocus approach.
我们提出了一种使用相板进行聚焦数据采集的方法，该方法可实现近原子分辨率的单颗粒重构。准确聚焦是获得高质量数据的决定因素。为了达到所需的精度，我们实施了双区域聚焦策略。通过这种方法，我们获得了嗜酸性热原体 20S 蛋白酶体的 3.2Å分辨率重构。相板的性能与传统离焦方法相当或略有提高。对于使用聚焦相板图像达到 3Å 以下分辨率的情况，球面像差成为限制因素。相板可以使传统离焦方法难以解决的小尺寸、异质性和灵活性等挑战性样本的单颗粒分析成为可能。
