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.

A new view on crystal harvesting.

X-ray crystallography typically requires the mounting of crystals, which can make the sample difficult to manipulate when it is small and the microscope objective is close to the crystallization plate. By simply moving the objective to the bottom of a clear crystallization plate (inverting the normal view), crystals were able to be manipulated and harvested from wells having a 0.9 mm diameter and 5.

The structure of yeast glutaminyl-tRNA synthetase and modeling of its interaction with tRNA.

Eukaryotic glutaminyl-tRNA synthetase (GlnRS) contains an appended N-terminal domain (NTD) whose precise function is unknown. Although GlnRS structures from two prokaryotic species are known, no eukaryotic GlnRS structure has been reported. Here we present the first crystallographic structure of yeast GlnRS, finding that the structure of the C-terminal domain is highly similar to Escherichia coli GlnRS but that 214 residues, including the NTD, are crystallographically disordered.

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-tRNA(Gln) is primarily synthesized by first forming Glu-tRNA(Gln), followed by conversion to Gln-tRNA(Gln) by a tRNA-dependent amidotransferase.

What's in a drop? Correlating observations and outcomes to guide macromolecular crystallization experiments.

Observations of crystallization experiments are classified as specific outcomes and integrated through a phase diagram to visualize solubility and thereby direct subsequent experiments. Specific examples are taken from our high-throughput crystallization laboratory which provided a broad scope of data from 20 million crystallization experiments on 12,500 different biological macromolecules. The methods and rationale are broadly and generally applicable in any crystallization laboratory.

Small angle X-ray scattering as a complementary tool for high-throughput structural studies.

Structural crystallography and nuclear magnetic resonance (NMR) spectroscopy are the predominant techniques for understanding the biological world on a molecular level. Crystallography is constrained by the ability to form a crystal that diffracts well and NMR is constrained to smaller proteins. Although powerful techniques, they leave many soluble, purified structurally uncharacterized protein samples.

Crystal cookery - using high-throughput technologies and the grocery store as a teaching tool.

Crystallography is a multidisciplinary field that links divergent areas of mathematics, science and engineering to provide knowledge of life on an atomic scale. Crystal growth, a key component of the field, is an ideal vehicle for education. Crystallization has been used with a 'grocery store chemistry' approach and linked to high-throughput remote-access screening technologies.

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.