9 research outputs found
The application and use of chemical space mapping to interpret crystallization screening results
Mapping crystallization results in chemical space helps to correlate seemingly distant relationships between crystallization conditions, points to possible optimization strategies and reveals promising unsampled areas of crystallization space
Crystal cookery – using high-throughput technologies and the grocery store as a teaching tool
Using high-throughput crystallization screening technologies and data analysis, an educational program has been developed to teach the scientific method through crystallization and access to a grocery store, a post office and the internet
Structural conservation of an ancient tRNA sensor in eukaryotic glutaminyl-tRNA synthetase
In all organisms, aminoacyl tRNA synthetases covalently attach amino acids to their cognate tRNAs. Many eukaryotic tRNA synthetases have acquired appended domains, whose origin, structure and function are poorly understood. The N-terminal appended domain (NTD) of glutaminyl-tRNA synthetase (GlnRS) is intriguing since GlnRS is primarily a eukaryotic enzyme, whereas in other kingdoms Gln-tRNAGln is primarily synthesized by first forming Glu-tRNAGln, followed by conversion to Gln-tRNAGln by a tRNA-dependent amidotransferase. We report a functional and structural analysis of the NTD of Saccharomyces cerevisiae GlnRS, Gln4. Yeast mutants lacking the NTD exhibit growth defects, and Gln4 lacking the NTD has reduced complementarity for tRNAGln and glutamine. The 187-amino acid Gln4 NTD, crystallized and solved at 2.3 Å resolution, consists of two subdomains, each exhibiting an extraordinary structural resemblance to adjacent tRNA specificity-determining domains in the GatB subunit of the GatCAB amidotransferase, which forms Gln-tRNAGln. These subdomains are connected by an apparent hinge comprised of conserved residues. Mutation of these amino acids produces Gln4 variants with reduced affinity for tRNAGln, consistent with a hinge-closing mechanism proposed for GatB recognition of tRNA. Our results suggest a possible origin and function of the NTD that would link the phylogenetically diverse mechanisms of Gln-tRNAGln synthesis
What’s in a Drop? Correlating Observations and Outcomes to Guide Macromolecular Crystallization Experiments
The detection and subsequent volume optimization of biological nanocrystals
Identifying and then optimizing initial crystallization conditions is a prerequisite for
macromolecular structure determination by crystallography. Improved technologies
enable data collection on crystals that are difficult if not impossible to detect using
visible imaging. The application of second-order nonlinear imaging of chiral crystals and
ultraviolet two-photon excited fluorescence detection is shown to be applicable in a
high-throughput manner to rapidly verify the presence of nanocrystals in crystallization
screening conditions. It is noted that the nanocrystals are rarely seen without also producing
microcrystals from other chemical conditions. A crystal volume optimization method is
described and associated with a phase diagram for crystallization
Efficient optimization of crystallization conditions by manipulation of drop volume ratio and temperature
An efficient optimization method for the crystallization of biological macromolecules has been developed and tested. This builds on a successful high-throughput technique for the determination of initial crystallization conditions. The optimization method takes an initial condition identified through screening and then varies the concentration of the macromolecule, precipitant, and the growth temperature in a systematic manner. The amount of sample and number of steps is minimized and no biochemical reformulation is required. In the current application a robotic liquid handling system enables high-throughput use, but the technique can easily be adapted in a nonautomated setting. This method has been applied successfully for the rapid optimization of crystallization conditions in nine representative cases