Abstract
In 2003, a fully automated protein crystallization and monitoring system (PXS) was developed to support the structural genomics projects that were initiated in the early 2000s. In PXS, crystallization plates were automatically set up using the vapor-diffusion method, transferred to incubators and automatically observed according to a pre-set schedule. The captured images of each crystallization drop could be monitored through the internet using a web browser. While the screening throughput of PXS was very high, the demands of users have gradually changed over the ensuing years. To study difficult proteins, it has become important to screen crystallization conditions using small amounts of proteins. Moreover, membrane proteins have become one of the main targets for X-ray crystallography. Therefore, to meet the evolving demands of users, PXS was upgraded to PXS2. In PXS2, the minimum volume of the dispenser is reduced to 0.1 µl to minimize the amount of sample, and the resolution of the captured images is increased to five million pixels in order to observe small crystallization drops in detail. In addition to the 20°C incubators, a 4°C incubator was installed in PXS2 because crystallization results may vary with temperature. To support membrane-protein crystallization, PXS2 includes a procedure for the bicelle method. In addition, the system supports a lipidic cubic phase (LCP) method that uses a film sandwich plate and that was specifically designed for PXS2. These improvements expand the applicability of PXS2, reducing the bottleneck of X-ray protein crystallography.
Highlights
Three-dimensional structural information on proteins and other biological macromolecules is important to elucidate the molecular mechanisms of biochemical processes in living organisms, and to accelerate drug-discovery processes
In 2003, we developed a fully automated protein crystallization and monitoring system (PXS) to achieve highthroughput crystallization screening for a structural genomics project (Hiraki et al, 2006)
The main improvements are a reduction in the amount of sample that is required for crystallization screening and the addition of new options for membrane-protein crystallization
Summary
Three-dimensional structural information on proteins and other biological macromolecules is important to elucidate the molecular mechanisms of biochemical processes in living organisms, and to accelerate drug-discovery processes. Recent advances in the single-particle analysis method using cryo-electron microscopy (cryo-EM) have allowed us to determine protein structures at subatomic resolution with small sample volumes without crystallization (Liao et al, 2013; Cheng, 2018). It is still challenging to determine the structures of small proteins with a molecular mass of less than 40 kDa by single-particle analysis using cryoEM. In pharmaceutical science, highthroughput drug screening using MX is still a critical method.
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