Data collection ribosome

We have shown that out of fifteen forms of three-dimensional crystals from ribosomal particles, grown so far in our laboratory, some appear suitable for crystallographic data collection when using synchrotron radiation at temperatures between 19 °C and —180 °C 50S subunits from H. marismortui., and from B. stearothermophilus, including the -BLl 1 mutant, and the new crystal forms from B. stearothermophilus SOS and Thermus thermophilus 30S subunits which have only recently been grown in non-volatile precipitants We also plan to continue research on biochemically modified particles, such as SOS with one tRNA and its nascent polypeptide chain (which have already been crystallized). 

Spectacular use of cryocrystallography has been made to avoid an impasse reached in data collection at or near room temperature from ribosome crystals (Hope et al 1989). At room temperature these samples decay in the X-ray beam (monochromatic X in the range 0.9-1.5A) dramatically making data collection beyond 18 A essentially impossible. At liquid nitrogen temperatures an initial resolution of 5 A is preserved essentially indefinitely. Mosaic spreads are unchanged between room temperature and cryotemperatures but are quite large anyway (—3°). The intensity of the synchrotron beam is used to make the data collection time manageable. 

The combination of all the advantages of SR is especially needed in virus and ribosome crystallography where the unit cells are very large. Data collection from very large unit cell constant crystals benefits from... 

The exploitation of this radiation, particularly the brilliance and use of short A s, has made virus crystal data collection routine from difficult samples although it is at present necessary to use hundreds of crystals in the gathering of just one data set. Maybe the use of ultra-short wavelength beams ( =0.33 A) from a harmonic of an undulator could be harnessed to improve the lifetime of one such sample sufficiently to give a complete data set. Much larger macromolecular assemblies are currently under study, such as the ribosome, which possess little or no symmetry (unlike viruses) and are therefore more difficult to solve. 

The synchrotron should not be seen as a replacement for facilities in the home laboratory but as a means for meeting technically challenging data collection problems. Of course, in the absence of any home X-ray facilities, the central facility can be used but this is not terribly efficient because of the necessity of long-term scheduling of many users. Hence, characterisation of samples (e.g. of heavy atom derivatives) should be done at home unless the project is entirely reliant on the SR source in this category are many virus studies and ribosome crystallography as well as small crystal projects. 

The ribosome has been a favorite nucleo-protein for study by neutron scattering (References in (3)). We shall mention briefly a recent experiment which made use of the scattering curve I(Q) and [H2O] [ D2O ] variation in the solvent (3). Electron micrographs of 16S RNA had been obtained and showed it to have a Y shaped structure. Is this structure maintained within the 30 S ribosome particle The scattering curve of the model was calculated and compared with neutron data. In 42% D2O, the excess scattering mass of protein is negligible, so that data collected from the 30 S in this buffer should only reflect the RNA structure within the particle. The... 

Ribosomes, the intracellular particles on which proteins are assembled, are highly complex and dynamic entities. The structural framework of ribosomes is provided by ribosomal RNA (rRNA) molecules with which many proteins are associated (summarized in Capowski and Tracy, 2003). Homologous rRNA genes occur in all prokaryotes and eukaryotes. The mitochondrial and chloroplast rRNA genes in eukaryotes clearly have prokaryote affinities (Pace et ai, 1986). The genomic DNA from which ribosomal genes are transcribed, along with any associated spacers, is collectively termed ribosomal DNA (rDNA). Sequences and other data from rDNA and its products,... 

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