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

1996年的项目方向是完成这三项工作 NCD和Kinesin运动域的空间结构。 1996年初，突变的激动素Gly234Ala， 它与微管紧密结合，但不水解三磷酸腺苷。这个 突变是一种普遍保守的甘氨酸，在同源基因中 肌球蛋白和G蛋白参与G蛋白的结合和感觉 三磷酸腺苷的磷酸。有关突变体的X射线数据是在CHESS收集的 束线F1(L=0.908)。与镁ADP结合的突变体显示没有 显著的结构变化(C.Sindelar，J.Kull等人， 正在准备中的手稿)。坐标正在改进中。我们 我还尝试将Kinesin和NCD的运动域结晶在 核苷酸诱导的交替构象状态(与ATPg-S， ADPNP或ADPCP绑定)。没有获得衍射级晶体 到目前为止关于这些核苷酸的激动素。NCD马达领域拥有 在AMPPCP存在下结晶，但晶体是 仍然太小，无法进行X射线衍射(20x10x10毫米) 已经被用于获得二聚体形式的运动蛋白晶体 和NCD具有不同的构象状态。晶体的获得是为了 NCD的三种不同结构，尽管它们都显示出弱 绕射。最佳数据(至4.5Rmerge 8.6%)被收集 用RAXIS II研究NCD二聚体D250-K700的速冻晶体 图像板探测器。晶体属于单斜晶系。 单胞尺寸为a=153.4的P21群，b=201.4，c=195.3和 B=90.5o。尝试通过以下方法获得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。分子置换解决方案适应 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诱导的交替核苷酸状态结晶。 这些晶体仍然太小(10x10x5毫米)，无法进行X射线检查 绕射。我们正在寻找新的晶体形式，通过 不同的条件或具有替代结构。

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.