HG-MAD DATA COLLECTION OF PF FIBRILLARIN-NOP5P COMPLEX

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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.