CRYSTALLOGRAPHIC STUDIES ON RIBOSOMES

核糖体的晶体学研究

• 批准号: 6120548
• 负责人: ADA E YONATH
• 金额: $ 0.02万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-15 至 1999-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6120548
• 关键词: bioimaging /biomedical imaging; biological products; biomedical resource; proteins; radiography; structural biology

Ribosomes, the universal cell organelles facilitating protein biosynthesis, are nucleoprotein assemblies (mw 2.3 MD), composed of 57-73 different proteins and 3 RNA chains of about 4500 nucleotides, arranged in two subunits of unequal size. Crystals, diffracting best to around 3
, have been grown fron intact large and from small ribosomal subunits (from haloarcula marismortui, H50S, and Thermus thermophilus, T30S, respectively). The latter are of high inteest since the small subunits are known to be extremely unstable and flexible and until recently yielded crystals diffracting to low resolution (7.3
at best, useful to 10-12
). Data were collected with Synchrotron raditation (at F1/CHESS) at cryogenic temperatures. The brightness necessary for the collection of the higher resolution X-ray diffraction data introduces significant decay even at cryo temperature. Nevertheless, owing to the reasonable isomorphism of the recently improved crystals of the small subunits, reliable phases (as well as an electron density map) have been extracted at medium (6.5
) resolution, using the combination of relatively large clusters with smaller metal compounds. The crystals of the large subunits are more problematic. It was found that their substantial radiation sensitivity accompanied by a low level of isomorphism, changes in unit cell axis (introduced by irradiation) and their non-isotropic mosaicity become more pronounced and therefore less tolerable with the increase in resolution, hampering smoot MIR phasing. Some of these problems were partially ovecome by using a beam of a rather small cross-section and irradiating the crystals part by part. Consequently, in favorable cases, up to 20 degrees were collected from one crystal. To gain insight on the structure of these crystals A 12
resolution MIR electron density map of H50S was constructed, using Ta6Br14, W3O2((acetate)6H2O)3)CFSO3, K6H(PW12O40)nH2O and Cs7(P2W17O61Co(NC5H5))nH2O for soaking. This map may indicate the reasons for the problematic nature of the crystals and provide hints on ways for their improvement.

核糖体，通用细胞细胞器促进蛋白质 生物合成是核蛋白组件（MW 2.3 MD），由 57-73不同的蛋白质和3个RNA链，约4500个核苷酸， 以两个不平等的亚基排列。 晶体，衍射最好 到3左右，已经成长为完整的大大，从小 核糖体亚基（来自Haloarcula Marismortui，H50S和Thermus Thermophilus，T30S）。 后者是最高的 由于小亚基非常不稳定，并且 柔性，直到最近产生的晶体衍射为低 分辨率（最多为7.3，可用于10-12）。 收集数据 在低温温度下的同步加速器辐射（在F1/国际象棋时）。 收集高分辨率所需的亮度 X射线衍射数据即使在低温下也会引入显着衰变 温度。 然而，由于合理的同构 最近改善了小亚基的晶体，可靠的相（AS 作为电子密度图）已在培养基处提取（6.5） 解决方案，使用相对较大的簇与 较小的金属化合物。 大亚基的晶体更多 有问题。 发现他们的辐射很大 敏感性伴随着低水平的同构，单位变化 细胞轴（通过辐射引入）及其非偏性 镶嵌变得更加明显，因此与 分辨率增加，阻碍了mir逐渐缩小。 其中一些 通过使用一束相当小的光束，问题部分是卵子的 横截面并部分辐照晶体。 因此，在有利的情况下，从中收集了多达20度 一个晶体。 为了了解这些晶体的结构12 使用H50S的分辨率miR电子密度图构建了 TA6BR14，W3O2（（乙酸）6H2O）3）CFSO3，K6H（PW12O40）NH2O和NH2O和 CS7（P2W17O61CO（NC5H5））NH2O浸泡。 该地图可能表明 晶体有问题的原因并提供提示 关于他们改进的方式。