Crystal identification and delivery technology for serial synchrotron crystallography

Atomic structures of biomolecules (e.g., proteins) and biomolecular complexes facilitate detailed understanding of biomolecular function. They are key drivers of the ongoing revolution in molecular biology and of many areas of biotechnology, including development of pharmaceuticals and of enzymes and light harvesters for bioenergy. Most biomolecular structures are determined by X-ray crystallography using crystallized biomolecules. Advances in synchrotron X-ray sources, especially at DOE laboratories, have reduced data collection times per crystal from tens of minutes to less than one second, and enabled useful data to be obtained from micrometer size crystals. Advances in structure determination methods using X-ray free-electron laser sources (especially the DOE’s LCLS) have driven worldwide efforts to develop serial synchrotron X-ray crystallography (SSX). In SSX, dozens to tens of thousands of crystals are sequentially irradiated, and data from these crystals merged to determine a biomolecule’s structure. Many sample delivery systems have been developed for SSX, but no one system is ideal for all SSX applications, and the diversity of approaches has hindered adoption of SSX methods. Identifying microcrystals on SSX sample holders remains challenging but is essential to maximize the efficiency of X-ray data collection and processing. This STTR project aims to develop a flexible, integrated, cost-effective platform for sample delivery and imaging in serial synchrotron X-ray crystallography. The sample delivery system incorporates elements from published designs and our innovations to accommodate a variety of real-world sample delivery needs and utilizes to the maximum extent possible the large existing infrastructure for sample handling at DOE laboratories. The imaging system draws on advances at Cornell University in the design of high peak power, femtosecond pulse laser sources and expertise in multimodal laser scanning microscopy that could allow large improvements in microcrystal imaging performance and speed while significantly lowering imaging system costs. In Phase I of this project, prototype sample holders and a sample loading station for serial synchrotron crystallography were designed, fabricated, and tested. Two different approaches to generating femtosecond pulse lasers were explored. A laser meeting our core performance specifications was developed and used to image microcrystals by two-photon excitation fluorescence and second harmonic generation. In Phase II of this project, the sample holders and sample loading station will be evolved to final commercial designs. Development of femtosecond pulse lasers based on the Phase I approaches will continue, focusing on obtaining robust, low cost designs suitable for commercialization. A multimodal laser scanned imaging system for imaging microcrystals on SSX sample holders, incorporating the developed laser, will be constructed. Software for automated recognition of microcrystals and generation of coordinates for efficient synchrotron data collection will be developed. These technologies will speed adoption of SSX methods, which will have a significant impact on structural studies of membrane proteins, large biomolecular machines, and other systems for which obtaining large crystals is challenging. SSX will also dramatically increase structure determination throughput, enabling studies of large numbers of variants of a given biomolecule as required, e.g., in functional optimization of bioenzymes, and of interactions of large numbers of ligands with a given biomolecule as required, e.g., in pharmaceutical development. The proposed work will thus support advances in medical treatments, pharmaceuticals, bioenergy, and biotechnology that will be key drivers of US economic growth in the coming decades. This project and the manufacture and sale of the products it generates will provide good paying jobs and support economic development in the Southern Tier of New York State, which includes many of the state’s most economically depressed counties and cities.