XRAY CRYSTALLOGRAPHY OF CATALYTIC RNA

催化 RNA 的 X 射线晶体学

• 批准号: 6120485
• 负责人: JENNIFER A DOUDNA
• 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/6120485
• 关键词: bioimaging /biomedical imaging; biological products; biomedical resource; proteins; radiography; structural biology

We are pursuing structure determination of a number of catalytic RNA molecules and catalytic RNA domains by X-ray crystallography. In the past several years we have developed novel methods to obtain diffraction quality crystals of large RNA molecules which employ "RNA crystallization modules". These are moieties engineered into the primary sequence of the target RNAs to present well-differentiated molecular surfaces that promote inter-molecular contacs, and crystal growth. Progress has been made in two fronts employing the high brilliance X-rays available at CHESS. 1) Crystals of a 70-nucleotide domain of a self-splcing Group II intron were obtained in a hexagonal space group (a=94 A, c=230 A) which diffracted weakly to 4 A resolution with a laboratory X-ray source. Optimization of flash cooling conditions, and data collection at beam-line A-1 of CHESS with the ADSC Quantum-1 area detector produced a complete native data-set to 3.4 A resolution. Preparation of heavy atom derivatives is underway. 2) Crystals of the complete catalytic core of the self-cleaving ribozyme from the genomic RNA of the human Hepatitis Delta Virus (HDV) which diffractedto 3.4 A in the laboratory, were obtained. The structure was solved by multiwavelength anomalous diffraction (MAD) employing synchrotron radiation from a bending magnet at the National Synchrotron Light Source (Brookhaven National Laboratory, NY) where data to 2.7 A resolution were obtained. Improved flash cooling and the high brilliance and collimation of X-rays available at beam-line F1 of CHESS allowed us to collect a superior native data-set which extends to beyond 2.4 A resolution on the ADSC Quantum-4 area detector. Refinement of the structure against this higher resolution data-set is in progress.

我们正在进行一些催化剂的结构测定， RNA分子和催化RNA结构域的X射线晶体学。 在 在过去的几年里，我们开发了新的方法来获得 使用“RNA”的大RNA分子的衍射质量晶体 结晶模块”。 这些是工程化到 靶RNA的一级序列，以呈现分化良好的 促进分子间接触的分子表面， 增长 在两个方面取得了进展， 在国际象棋中可以获得的X光片。 1)70个核苷酸的晶体 结构域的自我剪接组II内含子中获得的六边形 空间群（a=94 A，c=230 A），弱衍射至4 A 分辨率与实验室X射线源。 Flash优化 冷却条件，并在CHESS的光束线A-1处收集数据， ADSC Quantum-1区域探测器产生了一个完整的本地数据集 3.4A分辨率 制备重原子衍生物， 正在进行中 2)晶体的完整催化核心的 人肝炎病毒基因组RNA自切割核酶 在实验室中衍射至3.4A的Delta病毒（HDV）， 得到了 用多波长反常解析法求解了结构 衍射（MAD）采用来自弯曲的同步辐射， 国家同步加速器光源（Brookhaven National） 实验室，纽约），其中获得了2.7 A分辨率的数据。 改进的闪光冷却和高亮度和准直 在CHESS的光束线F1处可用的X射线允许我们收集 上级原生数据集，分辨率超过2.4 A， ADSC Quantum-4区域探测器 改进结构， 这一更高分辨率的数据集正在制作中。