ELECTRON MICROSCOPY OF MACROMOLECULAR STRUCTURES

提出的研究将改进高分辨率电子的方法。 大分子的显微镜，并将继续将其应用于 柯根生物问题。 用钨薄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、颗粒尺寸、 而不是工具因素，目前限制了最终的解决方案。 因此，我们打算生产细粒薄膜，这将充分利用 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，从而减小微晶尺寸)和低温 试片支架(应有利于胶片的连续性)。的效果 还将研究溶剂体系和干燥条件。这些 这些技术将应用于许多大分子系统，包括 仓鼠雌性蛋白，来自无脊椎动物的IgM，人类润滑剂，支气管 粘蛋白等。 我们计划电子显微镜测绘方法的应用，其中 大分子与易分辨的、特定部位的标记物形成络合物。 一系列的问题。纤维蛋白原上的化学定义的位置 分子，包括结合因子XIII的重要区域， 葡萄球菌素和血小板，将通过络合定位 特定定向的纤维蛋白或与单抗结合。的位置 这个分子中的碳水化合物残留量将通过络合来测定。 有特定的凝集素。骨骼肌球蛋白和心肌肌球蛋白将与 针对这些分子的亚区(S1、S2、Light)的特定Fabs 链&50kd和25kd的S1亚区)，以阐明其拓扑结构 肌球蛋白头部区域。碳水化合物残留物的位置 表观葡聚糖蛋白(一种小鼠乳腺肿瘤细胞表面糖蛋白)将 由一系列特异性凝集素和兔&单抗免疫球蛋白& 免疫球蛋白。后者将被用来确定这些 分别针对表葡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出来。 我们将用物理-化学和化学相结合的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 肌球蛋白及其多聚体的功能和结构的去除。

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.