Biophysical Chemistry ELSEVIER Biophysical Chemistry 50(1994) 3-16 Crystallography of halophilic ribosome: the isolation of an internal ribonucleoprotein complex

Crystals of SOS ribosomal subunits from Haloarcula rnatimortui diffracting to 2.9 A resolution were grown. Because of their large unit cells and the extremely weak diffracting power, almost all X-ray crystallographic analysis of these crystals must be performed with intense synchrotron radiation. At ambient emperature, all ribosomal crystals decay upon the first instance of X-irradiation. To overcome this severe sensitivity, procedures for data collection at cryo temperature were developed. Under these conditions the crystals can be irradiated for periods sufficient for the collection of more than one data set from an individual crystal (days or weeks) with no observable damage. They also can be stored for months, to resume interrupted measurements. To assist he interpretation f the anticipated electron density map, a specific internal nucleoprotein complex of protein HmaLl and a stretch of H23S rRNA was isolated from the halophilic ribosome. The fragments of the 23s rRNA protected by the protein from nuclease digestion were sequenced. Alignment of the sequences of some archaebacterial Ll-specific RNA fragments othe corresponding parts of eubacterial nd eukaryotic rDNAs, localized the sequence identities o two distinct regions. Chimeric omplexes were reconstituted with the corresponding E. cofi ribosomal components, indicating a rather high homology, despite the evolution distance. A feasible secondary structure of the rRNA stretch participating in this complex was found to be compatible with the one proposed for the corresponding part in the E. coli ribosmnal RNA. Key words: Haloarcula maristnortui; Ribosomes; X-ray crystallography; Ribonucleoprotein complexes 1