Cyclic Olefin Copolymer-Based Fixed-Target Sample Delivery Device for Protein X-ray Crystallography

Serial macromolecular X-ray crystallography plays an important role in elucidating protein structures and consequently progressing the field of targeted therapeutics. The use of pulsed beams at different repetition frequencies requires the development of various sample-conserving injection strategies to minimize sample wastage between X-ray exposures. Fixed-target sample delivery methods that use solid support to hold the crystals in the X-ray beam path are gaining interest as a sample-conserving delivery system for X-ray crystallography with high crystal hit rates. Here, we present a novel fixed-target microfluidic system for delivering protein microcrystals to X-ray beams for diffraction data collection and structure determination. The fixed-target design consists of 3 symmetric sections arranged in an area of 1 in. x 1 in. with up to 18,000 crystal traps per device. Each trap is targeted to hold one crystal up to 50 mu m in size in the largest dimension. The device has been fabricated using cyclic olefin copolymer (COC) for high-quality diffraction data collection with low background scattering induced through the fixed-target material. The newly developed fixed-target device is designed for vacuum compatibility which will enable the use in vacuum experimental chambers of X-ray radiation sources including the newly developed, first-of-its-kind compact X-ray light source (CXLS), which is currently in commissioning at Arizona State University. To assess the validity of the COC device, serial crystallography experiments were performed on the model protein lysozyme at the new European Synchrotron Radiation Facility-Extremely Brilliant Source (ESRF-EBS) beamline ID29. A 1.6 & Aring; crystal structure of the protein was solved, demonstrating that, in general, the COC device can be used to generate high-quality data from macromolecular crystals at the CXLS and synchrotron radiation sources, which holds enormous potential for advancing the field of protein structure determination by fixed-target X-ray crystallography.