Abstract
Determining optimal conditions for the production of well diffracting crystals is a key step in every biocrystallography project. Here, a microfluidic device is described that enables the production of crystals by counter-diffusion and their direct on-chip analysis by serial crystallography at room temperature. Nine 'non-model' and diverse biomacromolecules, including seven soluble proteins, a membrane protein and an RNA duplex, were crystallized and treated on-chip with a variety of standard techniques including micro-seeding, crystal soaking with ligands and crystal detection by fluorescence. Furthermore, the crystal structures of four proteins and an RNA were determined based on serial data collected on four synchrotron beamlines, demonstrating the general applicability of this multipurpose chip concept.
Highlights
Crystallography plays a central role in contemporary biology because it enables the visualization of the 3D architecture of biological macromolecules, which provides insights into their cellular functions and partnerships on the atomic scale (Giege & Sauter, 2010; Jaskolski et al, 2014)
Microfluidics was immediately regarded as a major breakthrough, especially for biochemists dealing with samples that are difficult to purify in large quantities, such as macromolecules from higher eukaryotes, large biological assemblies and membrane proteins (Hansen & Quake, 2003; van der Woerd et al, 2003)
We illustrate a range of applications for ChipX3 by the crystallization of seven soluble proteins, a membrane protein and an RNA duplex, as well as the structure determination of five ‘non-model’ macromolecules at room temperature using data collected on four beamlines at three different synchrotron sites
Summary
Crystallography plays a central role in contemporary biology because it enables the visualization of the 3D architecture of biological macromolecules, which provides insights into their cellular functions and partnerships on the atomic scale (Giege & Sauter, 2010; Jaskolski et al, 2014). Microfluidics was immediately regarded as a major breakthrough, especially for biochemists dealing with samples that are difficult to purify in large quantities, such as macromolecules from higher eukaryotes, large biological assemblies and membrane proteins (Hansen & Quake, 2003; van der Woerd et al, 2003) Despite their potential, microfluidic technologies have not yet been massively adopted by the global community for crystal growth, as illustrated by the limited number of PDB entries (only about 30 as of March 2019) that cite the use of microfluidic systems. We illustrate a range of applications for ChipX3 by the crystallization of seven soluble proteins, a membrane protein and an RNA duplex, as well as the structure determination of five ‘non-model’ macromolecules at room temperature using data collected on four beamlines at three different synchrotron sites This lab-on-a-chip approach simplifies and efficiently miniaturizes the crystallographic structure-determination process, from the sample to its 3D structure, in a single device. It offers a user-friendly, cost-effective solution for routine biocrystallographic investigations at room temperature
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