MICROSCOPY & IMAGE ANALYSIS OF UNSTAINED MACROMOLECULES

拟议的研究涉及冷冻电子显微镜的结构分析 以及对多种病毒的三维图像分析。 我们 近期目标是回答有关形态学的基本问题 和复杂生物大分子的组成。 沿着知识的获得 从生物化学、遗传学、免疫学和其他生物物理学研究， 我们的结构工作可以提供关于组装，稳定性， 以及大分子的其他功能特性。 我们的目标仍然是 主要侧重于大分子的结构研究， 太复杂了，用目前的晶体学技术无法检测。 的 我们的研究的广泛数量和范围是通过几个 富有成效的合作。 多种二十面体病毒的三维结构， 将检查亚病毒颗粒和病毒-抗体复合物。 这些 包括乳多空病毒科的成员（多瘤病毒，猿猴病毒40， 牛、人和棉尾兔乳头状瘤病毒），呼肠孤病毒科 （恒河猴轮状病毒和血清型-1呼肠孤病毒）、Comoviridae（豇豆花叶病毒 病毒[CPMV]）、四病毒科（Nudaurelia capensis beta和omega 病毒）和枯草芽孢杆菌的等轴前体突变体 噬菌体phi29。 方法研究自然（非自然） 将研究等距Φ 29头。 这些病毒都广泛地 在不同的生物学科下进行研究。 尽管 大量的信息，详细的结构知识， 这些病毒是有限的。 蛋白质和核酸成分的组织在每一个 分子将被研究，以及蛋白质-蛋白质和蛋白质- 核酸相互作用负责稳定性，组装， 复合物的分解。 化学计量和相对 多肽在这些大分子中的分布将通过 抗体标记和通过比较成熟颗粒的结构（例如， 完整的病毒体）与部分组装或分解的颗粒（例如， 中间亚病毒颗粒、核心颗粒、空衣壳等）。 CPMV原子结构的知识将指导我们对 冷冻电镜研究中的病毒中和机制 CPMV与Fab和IgG分子的复合物。 低温电子显微镜和图像分析方法将进一步发展 i）允许大的和高度复杂的分子的结构 二）使处理程序更加灵活和稳健 iii）用抗体定位病毒中和位点 和抗体片段标记，iv）检测核酸和蛋白质- 病毒粒子内的核酸组织，v）检查 电子辐射对冷冻水化试样的损害，vi）改善 重建密度图中的对比度和分辨率（例如通过校正 对于相衬转移效应，或者通过对非常大量的 粒子图像），以及vii）用模型对X射线晶体学数据进行相位分析 从显微照片重建的密度图中得出。

The proposed studies involve structural analyses by cryoelectron microscopy and three-dimensional image analysis of a wide range of viruses. Our immediate goals are to answer fundamental questions about the morphology and composition of complex biomacromolecules. Along with knowledge gained from biochemical, genetic, immunological, and other biophysical studies, our structural work can provide new insights about the assembly, stability, and other functional properties of macromolecules. Our aim continues to focus mainly on structural studies of macromolecules that are too large or too complex to examine with current crystallographic technology. The extensive number and scope of our studies is made possible through several fruitful collaborations. The three-dimensional structures of a wide variety of icosahedral viruses, subviral particles, and virus-antibody complexes will be examined. These include members of the Papovaviridae (polyoma virus, simian virus 40, and bovine, human, and cottontail rabbit papilloma viruses), the Reoviridae (rhesus rotavirus and serotype-1 reovirus), the Comoviridae (cowpea mosaic virus [CPMV]), the Tetraviridae (Nudaurelia capensis beta and omega viruses), and isometric prohead mutants of the Bacillus subtilis bacteriophage phi29. Methods to study the morphology of the native (non- isometric phi29 head will be investigated. These viruses are all widely studied under a diverse range of biological disciplines. Despite an immense wealth of information, detailed structural knowledge about many of these viruses is limited. The organization of protein and nucleic acid components in each of the molecules will be studied, as well as the protein-protein and protein- nucleic acid interactions responsible for the stability, assembly, and disassembly of the complexes. The stoichiometries and relative distributions of polypeptides in these macro-molecules will be examined by antibody labeling and by comparing the structures of mature particles (e.g. full virions) with partially assembled or disassembled particles (e.g. intermediate subviral particles, core particles, empty capsids, etc.). Knowledge of the atomic structure of CPMV will guide our examination of the mechanism of virus neutralization in the cryo-electron microscopy studies of complexes of CPMV with Fab and IgG molecules. The methods of cryoelectron microscopy and image analysis will be further developed i) to allow the structures of large and highly complex molecules to be studied, ii) to make processing procedures more flexible and robust as well as efficient, iii) to map virus neutralization sites with antibody and antibody-fragment labels, iv) to examine nucleic acid and protein- nucleic acid organization inside virions, v) to examine the effects of electron radiation damage on frozen-hydrated specimens, vi) to improve contrast and resolution in reconstructed density maps (e.g. by correcting for phase contrast transfer effects or by averaging very large numbers of particle images), and vii) to phase x-ray crystallographic data with models derived from density maps reconstructed from micrographs.