HG-MAD DATA COLLECTION OF PF FIBRILLARIN-NOP5P COMPLEX

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。强烈热球菌Nop 56/58-原纤蛋白复合物的结构测定：核糖体是蛋白质合成的主力，是一组复杂的核糖体RNA和蛋白质分子。从出生到成为成熟核糖体的关键组成部分，核糖体RNA被修剪，化学修饰，并可能被一类独特的酶折叠。小核糖核蛋白颗粒（snoRNP）指导核糖体RNA的位点特异性切割和化学修饰（2-O-甲基化和假尿苷化）。大多数C/D盒类型的snoRNP负责近100个位置的核糖体RNA的位点特异性2-O-甲基化。每个盒C/D snoRNP与四种蛋白质（Nop 56、Nop 58、原纤蛋白、15.5kD蛋白质）和盒C/D RNA组装。盒C/D RNA指导RNP酶与靶RNA的结合，而蛋白质含有实际的甲基化活性。三维结构形成了理解这种酶的分子机制的基础。我们已经结晶的同系物Nop 56/58（一个单一的同源物在Escherichia）在复杂的纤维蛋白，甲基转移酶。这两种蛋白质形成盒C/D snoRNP的保守核心。我们最初尝试用硒代蛋氨酸（se-met）多波长反常衍射确定结构是不成功的，由于穷人se-met晶体。然后进行使用含有半胱氨酸残基的重原子浸泡的突变蛋白（野生型蛋白不具有半胱氨酸）的第二策略。这需要对衍射数据进行广泛的筛选。虽然我们在筛选了八种不同的重原子浸泡的突变体后没有找到一种衍生物，但这促使我们转向第三种策略。联邦快递数据收集小组的努力对这一进程至关重要。该结构最终通过使用截断突变体来解决，该截断突变体促进了衍射超过2.5 μ m的se-met晶体的生长。该结构表明了snoRNP组装的诱导拟合的有趣可能性。描述这一结果的手稿目前正在编写中。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Structure determination of Pyrococcus furiosus Nop56/58-fibrillarin complex: Ribosomes, the workhorse of protein synthesis, are an intricate set of ribosomal RNA and protein molecules. From birth to being the key component of mature ribsome, ribosomal RNAs are trimmed, chemically modified and possibly folded by a unique class of enzymes. Small ribonucleoprotein particles (snoRNPs) direct site specific cleavage and chemical modifications (2¿¿-O-methylation and pseudouridylation) of ribosomal RNAs. The majority of box C/D type of snoRNPs is responsible for site specific 2¿¿-O-methylation of ribosomal RNAs at nearly 100 locations. Each box C/D snoRNP is assembled with four proteins (Nop56, Nop58, fibrillarin, 15.5 kD protein) and a box C/D RNA. The box C/D RNA directs binding of the RNP enzyme to the target RNA while the proteins contain the actual methylation activity. Three dimensional structures form the basis for understand the molecular mechanism of this enzyme. We have crystallized the homolog of Nop56/58 (a single homolog in Archaea) in complex with fibrillarin, the methyltransferases. These two proteins form the conserved core of the box C/D snoRNP. Our initial attempt for determining the structure using selenomethionine (se-met) multiple wavelength anomalous diffraction was not successful due to poor se-met crystals. A second strategy of using heavy-atom soaked mutant proteins containing cysteine residues (the wild-type proteins do not have cysteines) was then carried out. This required extensive screening of diffraction data. Although we did not find a single derivative after screening eight different mutants soaked with various heavy atoms, it prompted us to move on to a third strategy. The efforts by the NSLS FedEx data collection team were critical for this process. The structure was finally solved by using a truncation mutant which facilitated growth of se-met crystals that diffracted beyond 2.5 ¿¿ The structure suggests an interesting possibility of induced-fit of snoRNP assembly. The manuscript describing this result is currently in preparation.