HG-MAD DATA COLLECTION OF PF FIBRILLARIN-NOP5P COMPLEX

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。狂热杆菌Nop56/58-纤维蛋白复合体的结构测定：核糖体是蛋白质合成的主力，是一组错综复杂的核糖体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(SnoRNPs)直接对核糖体RNA进行位点特异性切割和化学修饰(2？-O-甲基化和假尿嘧啶)。大多数box C/D类型的snoRNPs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box C/D snoRNP由4种蛋白质(Nop56、Nop58、纤毛素，15.5kD蛋白质)和一个box C/D RNA组装而成。盒C/D RNA指导RNP酶与目标RNA的结合，而蛋白质包含实际的甲基化活性。三维结构为理解该酶的分子机制奠定了基础。我们已经将Nop56/58的同源物(在古生界中的单一同源物)结晶在与纤维蛋白的络合物-甲基转移酶中。这两个蛋白形成了box C/D snoRNP的保守核心。我们最初尝试用硒蛋氨酸(Se-Met)多波长反常衍射来确定结构，但由于Se-Met晶体很差，所以没有成功。第二种策略是使用含有半胱氨酸残基的重原子浸泡突变蛋白(野生型蛋白没有半胱氨酸)。这需要对衍射数据进行广泛的筛选。尽管在筛选了八个浸泡在各种重原子中的不同突变体后，我们没有发现一个衍生品，但这促使我们转向第三种策略。NSLS联邦快递数据收集团队的努力对这一进程至关重要。结构最终通过使用截断突变体来解决，该突变体促进了衍射率超过2.5°的Se-Met晶体的生长。该结构表明了一种有趣的可能性，即snoRNP组装的诱导匹配。描述这一结果的手稿目前正在准备中。

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.