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年的项目方向是完成了三个 NCD 和驱动蛋白运动域的维度结构。 1996 年初，获得了突变型驱动蛋白 Gly234Ala 的晶体， 它与微管紧密结合，但不水解 ATP。 这 突变是普遍保守的甘氨酸，在同系物中 肌球蛋白和 G 蛋白，参与 g 的结合和感应 ATP 的磷酸盐。 CHESS 收集了突变体的 X 射线数据 光束线 F1 (l=0.908 )。 结合 MgADP 的突变体没有表现出 显着的结构变化（C. Sindelar、J. Kull 等人， 手稿正在准备中）。 坐标正在完善中。 我们 还尝试将驱动蛋白和 NCD 的运动结构域结晶化 交替的、核苷酸诱导的构象状态（与 ATPg-S、 ADPNP 或 ADPCP 结合）。 没有获得衍射质量的晶体 到目前为止，对于具有这些核苷酸的驱动蛋白。 NCD 运动域有 在AMPPCP存在下结晶，但晶体是 对于 X 射线衍射来说仍然太小 (20 x 10 x 10 mm) 已致力于获得驱动蛋白二聚体形式的晶体 和NCD处于不同的构象状态。 获得晶体的目的是 非传染性疾病的三种不同结构，尽管它们都表现出较弱 衍射。 收集了最佳数据（Rmerge 为 4.5，为 8.6%） 使用 RAXIS II 在 NCD 二聚体 D250-K700 的闪冻晶体上 图像板探测器。 晶体属于单斜晶系 组 P21 的晶胞尺寸 a=153.4 、 b=201.4 、 c=195.3 和 b=90.5°。 尝试获得 NCD 二聚体的蛋白质相 碘修饰的核苷酸由于弱衍射而失败 晶体和碘。 分子替代解决方案以适应 二聚体结构不明确，但令人鼓舞。 NCD 二聚体是 还以 AMPPCP 诱导的替代核苷酸状态结晶。 这些晶体对于 X 射线来说仍然太小 (10 x 10 x 5 毫米) 衍射。 我们正在寻找根据以下方法获得的新晶体形式 不同的条件或替代的结构。