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

1996年项目方向已完成三个 NCD和驱动蛋白的运动域的三维结构。 1996年初，获得了突变驱动蛋白Gly 234 Ala的晶体， 它与微管紧密结合，但不水解ATP。 的 突变是普遍保守的甘氨酸，在同源物中， 肌球蛋白和G蛋白，参与G蛋白的结合和传感 ATP的磷酸盐 在CHESS收集了突变体的X射线数据 光束线F1（l=0.908
). 结合MgADP的突变体显示没有 重大结构性变化（C。 Sindelar，J. Kull等人， 手稿正在准备中）。 坐标正在修正中。 我们 还试图使驱动蛋白和NCD的马达结构域结晶， 交替的，核苷酸诱导的，构象状态（与ATPg-S， ADPNP或ADPCPbound）。 没有获得衍射质量的晶体 与这些核苷酸的结合。 NCD运动域具有 在AMPPCP存在下结晶，但晶体 对于X射线衍射来说仍然太小（20 x 10 x 10 mm） 已经涉及获得驱动蛋白的二聚体形式的晶体 和NCD处于不同的构象状态。 获得晶体用于 三种不同的NCD结构，尽管它们都表现出弱 衍射法 最佳数据（至4.5
与Rmerge 8.6%）收集 使用RAXIS II在NCD二聚体D250-K700的快速冷冻晶体上 成像板检测器 晶体属于单斜晶系 晶胞尺寸a=153.4的P21群
，B=201.4
，c=195.3
和 B= 90.5 °。 尝试获得NCD二聚体的蛋白质相， 碘修饰的核苷酸失败，因为从 晶体和碘。 分子替代解决方案， 二聚体结构是模糊的，虽然令人鼓舞。 NCD二聚体是 也在AMPPCP诱导的交替核苷酸状态下结晶。 这些晶体对于X射线来说仍然太小（10 x 10 x 5 mm） 衍射法 我们正在寻找新的晶体形式， 不同的条件或替代的结构。

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.