CO CRYSTALS OF HUMAN REPLICATION PROTEIN & SINGLE STRANDED DNA: CANCER

人类复制蛋白共晶

• 批准号: 6491115
• 负责人: ALED M EDWARDS
• 金额: $ 14.27万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-15 至 2002-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6491115
• 关键词: bioimaging /biomedical imaging; biological products; biomedical resource; proteins; radiography; structural biology

The project direction in 1996 has been the completion of the three dimensional structures of the motor domains of the NCD and kinesin. In early 1996, crystals were obtained for a mutant kinesin, Gly234Ala, which binds tightly to microtubules, but does not hydrolyze ATP. The mutation is of a universally conserved glycine that, in the homologs myosin and G proteins, participates in binding and sensing of the g phosphate of ATP. X-ray data on the mutant were collected at CHESS beam line F1 (l=0.908
). The mutant with MgADP bound shows no significant structural changes (C. Sindelar, J. Kull, et al., manuscript in preparation). The coordinates are being refined. We also attempted to crystallize the motor domains of kinesin and NCD in alternate, nucleotide-induced, conformational states (with ATPg-S, ADPNP or ADPCPbound). No diffraction quality crystals are obtained for kinesin with these nucleotides so far. The NCD motor domain has been crystallized in the presence of AMPPCP, but the crystals are still too small for X-ray diffraction (20 x 10 x 10 mm) Most effort has been directed to obtaining crystals of dimeric forms of kinesin and NCD in different conformational states. Crystals are obtained for three different constructs of NCD, though all of them show weak diffraction. The best data (to 4.5
with Rmerge 8.6%) were collected on a flash-frozen crystal of the NCD dimer D250-K700 using RAXIS II image plate detector. The crystals belong to the monoclinic space group P21 with unit cell dimensions a=153.4
, b=201.4
, c=195.3
and b=90.5o. Attempts to obtain protein phases for the NCD dimer with iodo-modified nucleotides failed because of weak diffraction from the crystals and iodines. Molecular replacement solutions to fit the dimer structure are ambiguous, though encouraging. The NCD dimer was also crystallized in an alternate nucleotide state induced by AMPPCP. These crystals are still too small (10 x 10 x 5 mm) for X-ray diffraction. We are searching for new crystals forms obtained under different conditions or with alternate constructs.

1996年的项目方向是三个的完成 NCD和驱动蛋白的运动结构域的尺寸结构。 1996年初，获得了突变驱动蛋白Gly234Ala的晶体， 与微管紧密结合，但不会水解ATP。 这 突变是一种普遍保守的甘氨酸，在同源物中 肌球蛋白和G蛋白参与G的结合和传感 ATP的磷酸盐。 在国际象棋时收集突变体的X射线数据 梁线F1（L = 0.908）。 具有MGADP结合的突变体显示NO 显着的结构变化（C. Sindelar，J。Kull等人， 手稿准备）。 坐标正在完善。 我们 还试图将驱动蛋白和NCD的运动域结晶 替代，核苷酸诱导的构象状态（带有ATPG-S， ADPNP或ADPCPBOUND）。 没有获得衍射质量晶体 到目前为止，对于这些核苷酸的驱动蛋白。 NCD电机域具有 在AMPPCP存在下结晶，但晶体是 对于X射线衍射（20 x 10 x 10毫米）仍然太小 已指示获取动力蛋白二聚体形式的晶体 和不同构象状态的NCD。 获得晶体 NCD的三种不同的结构，尽管它们都表现出薄弱 衍射。 收集了最佳数据（rmerge 8.6％） 使用Raxis II 图像板检测器。 晶体属于单斜空间 具有单元尺寸的组P21 a = 153.4，b = 201.4，c = 195.3和 B = 90.5O。 尝试获得NCD二聚体的蛋白质相 iodo修饰的核苷酸因弱衍射而失败 晶体和碘。 分子替代解决方案以适合 二聚体结构是模棱两可的，尽管令人鼓舞。 NCD二聚体是 也以AMPPCP诱导的替代核苷酸状态结晶。 这些晶体仍然太小（10 x 10 x 5 mm）X射线 衍射。 我们正在寻找在下面获得的新晶体形式 不同的条件或替代结构。