(789e) A Simplified Microseed Matrix Screening Method Using X-Ray Transparent High-Throughput Microfluidic Chips

Membrane proteins (MPs) are important targets for crystallization because they are transducers of material and signals across cell membranes and intracellular organelles, and their malfunction accounts for many diseases in humans.  One of the bottlenecks to obtaining diffraction quality crystals of membrane proteins is the need to screen thousands of crystallization conditions while negotiating their limited availability, instability in non-native lipids and detergents, and the long incubation times before the appearance of initial crystals.

A successful method to speed up the chances of finding the right crystallization conditions involves microseeding while setting up a matrix screening of the membrane protein.  However, microseed matrix screening involves disturbing the crystallization drop to introduce the microseeds by either a skill intensive and manually laborious procedure, or by microseeding robots. 

Here, we demonstrate a high-throughput X-ray compatible microfluidic platform for microseed matrix screening of crystallization conditions that enables rapid detection of crystallization conditions and optimization of microseeding protocols. The microfluidic chips, in 24-well and 96-well formats, allow for formulation of multiple microseed dilutions while consuming ~6 µL protein, precipitant and microseed solutions.  Our X-ray transparent chips enable room-temperature data collection while minimizing radiation damage by a strategy of collecting small wedges of data from multiple crystals and merging them to create a complete dataset. We validated our approach by crystallizing photoactive yellow protein and optimized the microseeding protocol to obtain large, single crystals. Further, we applied our method to investigate microseeding of bacterial photosynthetic reaction center, cytochrome c oxidase, and qNOR.