IVEM TOMOGRAPHY OF HIGH PRESSURE FROZEN & FREEZE SUBSTITUTED VIRUS STRUCTURES

(瑞典医学研究基金会MFR资助 瑞典战略研究基金会；瓦伦堡 研究基金会。P.I.R.H.Cheng)Cyro-EM，与 X射线结晶学数据和抗体标记，已经非常 有效地解决生物结构-功能问题。这个 IVEM有可能减少辐射损伤，更高 解决方案，也可以作为比较基础 对比度传递函数反褶积。几种类型的大型 病毒将通过冷冻-EM成像，包括倾斜的和非倾斜的。在……里面 此外，还将进行塑料切片层析成像研究 病毒萌发过程中的膜相互作用。在本报道期间 在此期间，重点放在萌芽过程和 病毒核心颗粒在细胞质和萌发过程中。BHK细胞 都感染了西姆利基森林病毒，高压 冷冻/冷冻替代，无化学固定。后染色 塑料部分0.25和0.12(M)的厚度使用 IVEM。较厚的部分被用来进行双倾斜 层析重建了一些萌芽中的病毒颗粒。 可以清楚地看到萌芽过程中的步骤，包括 当病毒颗粒到达细胞质时，它们会变平 膜，以及形成包膜的细节。一个 使用Sterecon进行分割，建立了表面渲染模型。这个 更薄的切片用于高倍率断层扫描 核心粒子的重建。核心粒子还没有被 从细胞中提取出来用于冷冻-EM，因此它们正在进行现场研究。 从两次双倾斜中重建了大约20个核心粒子 系列片。根据我们一直用来估算的标准 层析分辨率(相邻Z切片之间的相位残差)， 基于0.5 nm的像素大小，分辨率约为2 nm。我们是 将其与完整病毒颗粒内的核心颗粒进行比较 冷冻-EM后采用平均技术进行重建。修复 技术被用来优化重建，然后 平均化技术将与 断层扫描数据。为斯堪的纳维亚新兴市场撰写了一篇摘要 赫尔辛基社会会议：Kan，S.T.，Marko，M，Hultenby，K. 福赛尔，K.，加洛夫，H.，程，R.H.(1998)结构稳定性 甲型病毒的表面包膜和核衣壳核心。程序第50位 安。见见斯堪的纳维亚国家石油公司。电子显微镜E.L.普农宁和 E.Heikinheimo，编辑。PP。97-98。(

(Supported by Swedish Medical Research Foundation MFR K9706X1217501; Swedish Foundation for Strategic Research; Wallenberg Research Foundation. P.I. R.H. Cheng) Cyro-EM, in conjunction with X-ray crystallographic data and antibody labeling, has been very effective in addressing biological structure-function issues. The IVEM has the potential for decreased radiation damage, higher resolution, and also serves as a comparative base for contrast-transfer function deconvolution. Several types of large viruses will be imaged, both tilted and un-tilted, by cryo-EM. In addition, plastic-section tomography will be carried out to study membrane interactions during virus budding. During this reporting period, the focus was on the budding process and the structure of the virus core particle in the cytoplasm and during budding. BHK cells were infected with Simliki Forest virus, and high-pressure frozen/freeze-substituted without chemical fixation. Post-stained plastic sections 0.25 and 0.12 (m in thickness were examined using the IVEM. The thicker sections were used to make a double-tilt tomographic reconstruction of a number of budding virus particles. The steps in the budding process could be clearly seen, including flattening of the virus particles as they reached the cell plasma membrane, and the details of formation of the envelope. A surface-rendered model was made using Sterecon for segmentation. The thinner sections were used for high-magnification tomographic reconstructions of the core particles. Core particles have not been extracted from cells for cryo-EM, so they are being studied in-situ. About 20 core particles were reconstructed from two double-tilt series. According to criteria we have been using for estimating tomographic resolution (phase residuals between neighboring z-slices), the resolution was about 2nm, based on a pixel size of 0.5nm. We are comparing this to the core particles inside complete virus particles reconstructed using averaging techniques after cryo-EM. Restoration techniques are being used to optimize the reconstructions, then averaging techniques will be applied in combination with the tomographic data. An abstract was written for the Scandinavian EM society meeting in Helsinki: Kan, S.T., Marko, M, Hultenby, K., Forsell, K., Garoff, H., Cheng, R.H. (1998) Structural stability of surface envelope and nucleocapsid core of alphaviruses. Proc. 50th Ann. Meet Scandinavian Soc. for Electron Microscopy E.L Punnonen and E. Heikinheimo, Eds. pp. 97-98. (