ELECTRON MICROSCOPY OF MACROMOLECULAR STRUCTURES

这项研究将改善高分辨率电子测量的方法。 显微镜的大分子，并将追求这些应用， 共生生物学问题。 薄钨膜涂层有效地对比了大分子 下部结构 由于最近在电子光学、晶粒尺寸， 而不是工具性因素，目前限制了最终的解决办法。 因此，我们打算生产细颗粒薄膜， 可用的分辨率，并将适合于固有的 高对比度暗场成像模式。 为此，我们将探索使用 难熔金属的混合物（这应该限制 结晶，从而降低微晶尺寸）和低温 样品支撑（应有利于胶片连续性）。 的影响 还将研究溶剂系统和干燥条件。 这些 技术将应用于许多大分子系统，包括 仓鼠雌性蛋白，无脊椎动物IgM，人润滑素，支气管 粘蛋白等。 我们计划应用电子显微镜映射方法，其中 大分子与易于分辨的位点特异性标记物复合 一系列的问题。 纤维蛋白原上的化学定义位点 分子，包括结合因子XIII的重要区域， 葡萄球菌素和血小板，将通过与 特异性定向的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络合 特异性凝集素。 骨骼肌和心肌肌球蛋白将用 针对这些分子的亚区的特异性Fab（S1，S2，轻链 链和50 kd和25 kd S1亚结构域），以阐明 肌球蛋白头部。 糖残基的位置沿着 表聚糖蛋白（一种小鼠乳腺肿瘤细胞表面糖蛋白）将被 由一系列特异性凝集素绘制，并由兔和单克隆IgG和 IgM抗体 后者将用于确定这些的特异性 分别针对表聚糖蛋白和单特异性位点的抗体。 某些其他大的糖蛋白也将被绘制。 我们将结合物理化学和生物学方法来表征心肌肌球蛋白， 电子显微镜研究，以及评估轻链的影响 去除肌球蛋白及其聚合形式的功能和结构。

The proposed research will improve methods for high-resolution electron microscopy of macromolecules, and will pursue applications of these to cogen biological problems. Coating by thin tungsten films effectively contrasts 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 films which will fully exploit available resolving power, and which will be suited to the inherently high-contrast darkfield imaging mode. To this end we will explore use of mixtures of refractory metals (which should limit possibilities for crystalization and thus reduce crystallite size) and of low-temperature specimens 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 hamster female protein, IgM from invertebrates, human lubricin, bronchial mucins etc. We plan applications of the electronmicroscopic mapping approach, in which macromolecules are complexed with readily-resolvable, site-specific markers to a number of problems. Chemically-defined sites on the fibrinogen molecule, including the important region which binds factor XIII, staphylococcin & 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. Skeletal and cardiac myosin will be mapped with specific Fabs directed against subregions of these molecules (S1, S2, light chains & 50 kd & 25kd S1 subdomains) in order to elucidate topography of the myosin head region. Positions of carbohydrate residues along epiglycanin (a mouse mammary tumor cell-surface glycoprotein) will be mapped by a series of specific lectins, and by rabbit & monoclonal IgG & 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. We will characterize cardiac myosin in a combined physical-chemical and electron microscopic study, as well as assessing the effects of light chain removal on function and structure of myosin and its polymeric forms.