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

Area of Science:

• Structural Biology
• Biophysics
• Materials Science

Background:

• Serial macromolecular X-ray crystallography is crucial for determining protein structures and advancing targeted therapeutics.
• Pulsed X-ray sources necessitate sample-conserving injection strategies to minimize waste.
• Fixed-target delivery systems are emerging as efficient methods for X-ray crystallography, offering high crystal hit rates.

Purpose of the Study:

• To present a novel fixed-target microfluidic system for protein microcrystal delivery in X-ray crystallography.
• To demonstrate the system's capability for high-quality diffraction data collection and structure determination.
• To assess the device's compatibility with vacuum environments and new X-ray sources.

Main Methods:

• Fabrication of a fixed-target microfluidic device using cyclic olefin copolymer (COC) with up to 18,000 crystal traps per device.
• Delivery of protein microcrystals (up to 50 μm) to X-ray beams for diffraction data collection.
• Serial crystallography experiments conducted on lysozyme at the European Synchrotron Radiation Facility-Extremely Brilliant Source (ESRF-EBS) beamline ID29.

Main Results:

• A 1.6 Å crystal structure of lysozyme was successfully solved using the COC device.
• The device demonstrated high-quality data generation from macromolecular crystals.
• The fixed-target design is vacuum-compatible, suitable for use with compact X-ray light sources (CXLS).

Conclusions:

• The novel COC fixed-target microfluidic system is effective for serial X-ray crystallography.
• This technology holds significant potential for advancing protein structure determination.
• The system is compatible with current and future X-ray radiation sources, including CXLS.