CRYSTALLOGRAPHIC STUDIES OF REGULATED MYOSINS: MUSCLE CONTRACTION

在过去的一年里，杜立德和他的同事们已经确定了 人末端片段D结构域(M.W.86 kDa)和 从纤维蛋白获得的交联型D-D片段(Spraggon等， 1997年)。使用分子置换技术(使用X射线数据 在布鲁克海文和国际象棋上都收集了)，我们成功地 将片段D结构域定位在 闪速冷冻(和4C)完整牛纤维蛋白原晶体 分子。我们发现两人之间的端到端接触 我们晶体中与对称性有关的分子与已发现的相同 由杜利特尔和他的同事在人类D-D二聚体中发现。使用电子 密度差图，我们清楚地看到螺旋线圈区域连接 分子的末端D区和中心E区，现在可以 找到这些盘绕线圈中的弯头。这些弯道看起来是 分子柔韧性的主要特点。使用各种不同的 技术，包括改进的数据处理程序、改进、 和密度修改，我们现在正在延长分辨率 电子密度图至3.5(目前闪存冻结的限制 数据集)。我们现在正在追踪片段E和 连接线圈的区域，并处于第一个门槛 几乎整个纤维蛋白原的近原子分辨率图像 分子(Brown等人，正在准备中)。使用提供给我们的蛋白质 由L.Medve，我们还结晶了E的中心片段 牛纤维蛋白原(由二硫键稳定的 连接两个短螺旋线圈的球状区域)。数据增长至2.8 在国际象棋比赛中，从一个土生土长的人和7个重量级人那里收集到了解决方案 金属浸泡的碎片E晶体。在试图解开这个水晶时 结构，我们还使用从我们的 整个分子的结果。这一信息对于 了解纤维蛋白原在血液凝块中的组装 建立全面合理的药物设计方法 凝血障碍的治疗。

In the past year, Doolittle and colleagues have determined the structures of the terminal human fragment D domain (M.W. 86 kDa) and the crosslinked D-D fragment obtained from fibrin (Spraggon et al., 1997). Using molecular replacement techniques (with X-ray data collected both at the Brookhaven and at CHESS), we have succeeded in positioning the fragment D domains in the unit cells of the flash-frozen (as well as 4 C) crystals of the intact bovine fibrinogen molecule. We find that the end-to-end contacts made between symmetry-related molecules in our crystals are the same as those found by Doolittle and colleagues in the human D-D dimer. Using electron density difference maps, we clearly see coiled-coil regions connecting the terminal D and central E regions of the molecule, and can now locate the bends in these coiled coils. These bends appear to be the primary points of flexibility of the molecule. Using a variety of techniques, including improved data processing programs, refinement, and density modification, we are now extending the resolution of the electron density map to 3.5
(the present limit of the flash-frozen data sets). We are now tracing the chains in the Fragment E and connecting coiled coil regions, and are on the threshold of the first near-atomic resolution picture of virtually the whole fibrinogen molecule (Brown et al., in preparation). Using protein supplied to us by L. Medved, we have also crystallized the central Fragment E of bovine fibrinogen (which consists of a disulphide bridge-stabilized globular domain connecting two short coiled coils). Data to 2.8
resolution have been collected at CHESS from a native and 7 heavy metal-soaked Fragment E crystals. In attempting to solve this crystal structure, we are also using models of Fragment E derived from our results on the whole molecule. This information is vital for understanding the assembly of fibrinogen into the blood clot and for establishing a comprehensive rational drug design approach for treatment of clotting disorders.