ELECTRON MICROSCOPY OF MACROMOLECULAR STRUCTURES

该研究将改进高分辨率EM的方法， 单个大分子，并将追求这些应用到令人信服的 生物问题。 薄难熔金属膜涂层有效地对比了 大分子亚结构 由于最近电子技术的改进， 光学，粒度，而不是仪器因素，目前限制 终极解决方案 因此，我们打算生产细颗粒涂层， 将充分利用现有的解决能力，特别是应用 高对比度暗场成像。 我们将探索使用混合物 难熔金属（应限制结晶的可能性， 从而减小微晶尺寸）和低温样品支撑 （这应该有利于电影的连续性）。 溶剂系统的影响， 还将研究干燥条件。 这些技术将应用于 许多大分子系统，包括肌球蛋白，纤维蛋白原， 表多糖，视黄醇结合蛋白，抗原抗体复合物，支气管 粘蛋白和仓鼠雌性蛋白。 我们计划应用电子显微镜映射方法，其中 大分子与易于分解的位点特异性蛋白质复合 标记，一些问题。 化学定义的位点， 纤维蛋白原分子，包括结合因子的重要区域 高聚生与血小板，将通过络合定位 用特异性定向Fab或用单克隆抗体。 位置 该分子中的4个碳水化合物残基将通过以下方法确定： 与特异性凝集素复合。 我们将进一步描述心脏 肌球蛋白的物理化学和电子显微镜研究， 以及评估各种生理条件对 肌球蛋白S1区的功能和结构，以及聚合形式。 心脏 肌球蛋白将与针对肌球蛋白亚区的特异性Fab作图。 这些分子（S1、S2、轻链、S1亚结构域和铰链）， 阐明肌球蛋白头部区域的地形。 具体位置 表位和碳水化合物残基沿着表聚糖蛋白（一种小鼠乳腺肿瘤 细胞表面糖蛋白）将被一系列特异性凝集素定位， 以及兔和单克隆IgG和IgM。 后者将用于 确定这些抗体的特异性epiglycanin，和 单特异性位点。 某些其他大糖蛋白 也将被映射。

The proposed research will improve methods for high-resolution EM of individual macromolecules, and will pursue applications of these to cogent biological problems. Coating by thin refractory metal films effectively contrasts individual macromolecular substructure. Due to recent improvements in electron optics, grain size, rather than instrumental factors, currently limits ultimate resolution. Thus, we intend to produce fine-grain coatings which will fully exploit available resolving power, particularly applying high-contrast darkfield imaging. We will explore use of mixtures of refractory metals (which should limit possibilities for crystallization and thus reduce crystallite size) and of low temperature specimen supports (which should favor film continuity). Effects of solvent systems and drying conditions will also be studied. These techniques will be applied to a number of macromolecular systems, including myosin, fibrinogen, epiglycanin, retinol-binding protein, antigen-antibody complexes, bronchial mucins, and hamster female protein. We plan applications of the electronmicroscopic mapping approach, in which macromolecules are complexed with readily-resolvable, site-specific protein markers, to a number of problems. Chemically-defined sites in the fibrinogen molecule, including the important region which binds factor XIII, staphylococcin and blood platelets, will be located by complexing with specifically-directed Fabs or with monoclonal antibodies. Positions of the 4 carbohydrate residues in this molecule will be determined by complexing with specific lectins. We will further characterize cardiac myosin in a combined physical-chemical and electron microscopic study, as well as assessing the effects of various physiological conditions on function and structure of myosin S1 region, and polymeric forms. Cardiac myosin will be mapped with specific Fabs directed against subregions of these molecules (S1, S2, light chains, S1 subdomains and hinge) in order to elucidate topography of the myosin head region. Positions of specific epitopes and carbohydrate residues along epiglycanin (a mouse mammary tumor cell-surface glycoprotein) will be mapped by a series of specific lectins, and by rabbit and monoclonal IgG and IgM. The latter will be used to determine the specificities of these antibodies for epiglycanin, and for the monospecific site, respectively. Certain other large glycoproteins will also be mapped.