CRYSTALLOGRAPHIC STUDIES OF REGULATED MYOSINS: MUSCLE CONTRACTION

在过去的一年里，杜利特尔和他的同事们已经确定了 末端人片段D结构域的结构（M.W. 86 kDa）和 从纤维蛋白获得的交联D-D片段（Spraggon等， 1997年）。 使用分子置换技术（使用X射线数据 收集在布鲁克海文和国际象棋），我们已经成功地 将所述片段D结构域定位在所述细胞的晶胞中， 快速冷冻（以及4 ℃）的完整牛纤维蛋白原晶体 分子。 我们发现， 我们的晶体中与癌症相关的分子与发现的相同 由Doolittle及其同事在人类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和中心E区域，现在可以 找到这些螺旋的弯曲处 这些弯曲似乎是 分子的主要柔性点。 使用各种 技术，包括改进的数据处理程序，细化， 和密度修改，我们现在正在扩展的分辨率 电子密度图至3.5
(the速冻极限 数据集）。 我们现在正在追踪碎片E中的链条， 连接卷曲的线圈区域，并且在第一个的阈值上， 几乎整个纤维蛋白原的近原子分辨率图像 分子（Brown等人，正在筹备中）。 利用提供给我们的蛋白质 由L. Medved，我们还结晶了 牛纤维蛋白原（其由二硫桥稳定的 连接两个短卷曲螺旋的球状结构域）。 数据更新至2.8
决议已收集在国际象棋从一个本地和7重 金属浸泡的碎片E晶体。 在试图破解这块水晶的过程中 结构，我们还使用来自我们的片段E模型 整个分子的结果。 这些信息对于 了解纤维蛋白原组装成血凝块， 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.