(20e) Microfluidic Platforms for Crystallization Screening and In Situ Phasing & Structure Determination of Proteins

An ideal crystallization platform should be able to screen hundreds of crystallization conditions, allow for optimization of a successful hit which gives diffraction quality crystals and enable in situ structure determination from data collected on the platform preferably without having to harvest the crystal.

Our X-ray compatible, array-based microfluidic chip functions as a complete structure determination platform. To ensure X-ray compatibility we have eliminated as far as possible the use of polydimethylsiloxane (PDMS) which attenuates X-rays because of its silicon content. Instead our hybrid chip consists of different layers of materials: cyclic olefin copolymer (COC) for the valve layer, a thin, flexible PDMS fluid layer and a COC/Duralar substrate. The PDMS layer is needed for valve actuation to manipulate fluid flow on-chip. The scattering of X-rays by PDMS and the COC/Duralar material is minimal and does not affect the diffraction data.

The use of X-ray compatible materials in the chip eliminates manual harvesting of crystals thus allowing in situ analysis. This prevents damage to fragile protein crystals which might occur during harvesting. We have validated this by crystallizing on-chip lysozyme, thaumatin and ribonuclease A. We take advantage of the small scale and fluid handling capabilities of multilayer microfluidics to screen multiple crystallization conditions using minimal amount of protein (<2µL for 96 conditions). Once a successful condition is identified, the array based design allows growing multiple crystals on a single chip, following which slices of data can be collected from each crystal and later be merged together to give the complete data set.

To solve the structure of novel proteins it is essential to get phase information. Single wavelength anomalous dispersion (SAD) is one of the methods used for phasing to solve a protein structure. Our microfluidic platform allows the accurate measurement of the weak, anomalous diffraction differences between the heavy atoms (Selenium) used for phasing and the protein itself. We have successfully determined the structure of a novel bacterial lyase, PhNA to 2.3Å resolution using SAD phasing, solely using data collected on chip.