Stewart P L, Cary R B, Peterson S R, Chiu C Y
Department of Molecular and Medical Pharmacology and Crump Institute for Biological Imaging, UCLA School of Medicine, Los Angeles, California 90095, USA.
Microsc Res Tech. 2000 May 1;49(3):224-32. doi: 10.1002/(SICI)1097-0029(20000501)49:3<224::AID-JEMT2>3.0.CO;2-0.
Several advantages and disadvantages have been cited for image collection with a slow-scan CCD camera. Here we explore its use for cryo-EM single particle reconstruction and present two practical examples. The icosahedral adenovirus (Ad) type 2 ( approximately 150 MDa) was reconstructed from 396 particle images. The Fourier shell correlation (FSC) 0.5 threshold and the Fourier shell phase residual (FSPR) 45 degrees criterion yielded 17 AA resolution for the ordered viral capsid. Visual comparison with the filtered Ad2 crystallographic hexon confirmed a resolution range of 15-17 A. The asymmetric DNA-PKcs protein (470 kDa) was reconstructed from 9,473 particle images, using a previously published reconstruction based on class-sum images as an orientational search model [Chiu et al. (1998) J. Mol. Biol. 284:1075-1081]. FSC and FSPR methods yielded 17 A resolution for the new DNA-PKcs reconstruction, indicating a small but noticeable improvement over that of the class-sum based reconstruction. Despite the lack of symmetry for DNA-PKcs and its lower image contrast compared to Ad2 (0.8% vs. 2.5%), the same resolution was obtained for both particles by averaging significantly more DNA-PKcs images. Use of the CCD camera enables the microscopist to adjust the electron beam strength interactively and thereby maximize the image contrast for beam sensitive samples. On-line Fourier transformation also allows routine monitoring of drift and astigmatism during image collection, resulting in a high percentage of micrographs suitable for image processing. In conclusion, our results show that digital image collection with the YAG-scintillator slow-scan CCD camera is a viable approach for 3D reconstruction of both symmetric and asymmetric particles.
关于使用慢扫描电荷耦合器件（CCD）相机进行图像采集，人们已经列举了一些优缺点。在此，我们探索其在冷冻电子显微镜单颗粒重建中的应用，并给出两个实际例子。从396张颗粒图像重建了二十面体2型腺病毒（Ad）（约150 MDa）。傅里叶壳层相关（FSC）0.5阈值和傅里叶壳层相位残余（FSPR）45度标准得出有序病毒衣壳的分辨率为17埃。与经过滤波的Ad2晶体学六邻体进行视觉比较，确认分辨率范围为15 - 17埃。不对称的DNA依赖蛋白激酶催化亚基（DNA-PKcs）蛋白（470 kDa）是从9473张颗粒图像重建的，使用先前基于类和图像发表的重建结果作为取向搜索模型[邱等人（1998年）《分子生物学杂志》284:1075 - 1081]。FSC和FSPR方法得出新的DNA-PKcs重建的分辨率为17埃，表明相较于基于类和的重建有小幅但明显的改进。尽管DNA-PKcs缺乏对称性且与Ad2相比图像对比度较低（0.8%对2.5%），但通过平均更多的DNA-PKcs图像，两种颗粒都获得了相同的分辨率。使用CCD相机使显微镜工作者能够交互式地调整电子束强度，从而为对电子束敏感的样品最大化图像对比度。在线傅里叶变换还允许在图像采集过程中常规监测漂移和像散，从而得到高比例适合图像处理的显微照片。总之，我们的结果表明，使用YAG闪烁体慢扫描CCD相机进行数字图像采集是对称和不对称颗粒三维重建的一种可行方法。
