MICROSCOPY & IMAGE ANALYSIS OF UNSTAINED MACROMOLECULES

建议的研究包括用低温电子显微镜进行结构分析。 以及对多种病毒的三维图像分析。我们的 眼下的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。伴随着获得的知识 来自生化、遗传、免疫学和其他生物物理研究， 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性质。我们的目标是继续 主要关注过大或过大的大分子的结构研究 太复杂了，无法用当前的结晶学技术进行检查。这个 我们广泛的研究数量和范围是通过几个 卓有成效的合作。 各种二十面体病毒的三维结构， 将检查亚病毒颗粒和病毒-抗体复合体。这些 包括鹦鹉病毒科成员(多瘤病毒、猿猴病毒40和 牛、人和棉尾兔乳头瘤病毒)，呼肠孤病毒科 (恒河猴轮状病毒和1型呼肠孤病毒)，Comoviridae(豌豆花叶病毒 病毒[CPMV])，四联病毒科(Nudaurela capensis beta和omega 病毒)和枯草芽孢杆菌等长前部突变体 噬菌体phi29。方法对原住民(非原住民)进行形态观察 将对等轴测phi29头部进行调查。这些病毒都是广泛存在的 在不同的生物学学科下学习。尽管有一个 海量的信息，关于许多 这些病毒是有限的。 蛋白质和核酸成分的组织 将研究分子，以及蛋白质-蛋白质和蛋白质- 核酸相互作用负责稳定性、组装和 拆解复合体。化学计量学及其相关 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 完整的病毒粒子)具有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)。 对CPMV原子结构的了解将指导我们对 冷冻电子显微镜研究中的病毒中和机制 CPMV与Fab和Ig G分子的络合物。 低温电子显微镜和图像分析的方法将进一步发展 开发i)允许大分子和高度复杂的分子的结构 有待研究，二)使处理程序更加灵活和稳健 以及有效的，iii)用抗体定位病毒中和部位 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)以检查 冷冻水化试件的电子辐射损伤，vi)改善 重建密度图中的对比度和分辨率(例如，通过校正 对于相位对比度传递效果，或通过对非常大量的 粒子图像)，以及vii)将x射线结晶学数据与模型相关联 从显微照片重建的密度图派生而来。

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.