Reaching the potential of electron diffraction.

Microcrystal electron diffraction (MicroED) is an emerging structural technique in which submicron crystals are used to generate diffraction data for structural studies. Structures allow for the study of molecular-level architecture and drive hypotheses about modes of action, mechanisms, dynamics, and interactions with other molecules. Combining cryoelectron microscopy (cryo-EM) instrumentation with crystallographic techniques, MicroED has led to three-dimensional structural models of small molecules, peptides, and proteins and has generated tremendous interest due to its ability to use vanishingly small crystals. In this perspective, we describe the current state of the field for MicroED methodologies, including making and detecting crystals of the appropriate size for the technique, as well as ways to best handle and characterize these crystals. Our perspective provides insight into ways to unlock the full range of potential for MicroED to access previously intractable samples and describes areas of future development.

20 years of crystal hits: progress and promise in ultrahigh-throughput crystallization screening.

Diffraction-based structural methods contribute a large fraction of the biomolecular structural models available, providing a critical understanding of macromolecular architecture. These methods require crystallization of the target molecule, which remains a primary bottleneck in crystal-based structure determination. The National High-Throughput Crystallization Center at Hauptman-Woodward Medical Research Institute has focused on overcoming obstacles to crystallization through a combination of robotics-enabled high-throughput screening and advanced imaging to increase the success of finding crystallization conditions. This paper will describe the lessons learned from over 20 years of operation of our high-throughput crystallization services. The current experimental pipelines, instrumentation, imaging capabilities and software for image viewing and crystal scoring are detailed. New developments in the field and opportunities for further improvements in biomolecular crystallization are reflected on.

High-Throughput Screening to Obtain Crystal Hits for Protein Crystallography.

X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into an ordered lattice amenable to diffraction remains challenging. The crystallization of biomolecules is largely experimentally defined, and this process can be labor-intensive and prohibitive to researchers at resource-limited institutions. At the National High-Throughput Crystallization (HTX) Center, highly reproducible methods have been implemented to facilitate crystal growth, including an automated high-throughput 1,536-well microbatch-under-oil plate setup designed to sample a wide breadth of crystallization parameters. Plates are monitored using state-of-the-art imaging modalities over the course of 6 weeks to provide insight into crystal growth, as well as to accurately distinguish valuable crystal hits. Furthermore, the implementation of a trained artificial intelligence scoring algorithm for identifying crystal hits, coupled with an open-source, user-friendly interface for viewing experimental images, streamlines the process of analyzing crystal growth images. Here, the key procedures and instrumentation are described for the preparation of the cocktails and crystallization plates, imaging the plates, and identifying hits in a way that ensures reproducibility and increases the likelihood of successful crystallization.

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. 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 tRNA(Gln) 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-tRNA(Gln). These subdomains are connected by an apparent hinge comprised of conserved residues. Mutation of these amino acids produces Gln4 variants with reduced affinity for tRNA(Gln), 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-tRNA(Gln) synthesis.

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. This approach provides an educational opportunity that can effectively teach the scientific method, readily accommodate different levels of educational experience, and reach any student with access to a grocery store, a post office and the internet. This paper describes the formation of the program through the students who helped develop and prototype the procedures. A summary is presented of the analysis and preliminary results and a description given of how the program could be linked with other aspects of crystallography. This approach has the potential to bridge the gap between students in remote locations and with limited funding, and access to scientific resources, providing students with an international-level research experience.