The plug-based nanovolume Microcapillary Protein Crystallization System (MPCS)

Abstract

The Microcapillary Protein Crystallization System (MPCS) embodies a new semi-automated plug-based crystallization technology which enables nanolitre-volume screening of crystallization conditions in a plasticware format that allows crystals to be easily removed for traditional cryoprotection and X-ray diffraction data collection. Protein crystals grown in these plastic devices can be directly subjected to in situ X-ray diffraction studies. The MPCS integrates the formulation of crystallization cocktails with the preparation of the crystallization experiments. Within microfluidic Teflon tubing or the microfluidic circuitry of a plastic CrystalCard, $10-20 nl volume droplets are generated, each representing a microbatch-style crystallization experiment with a different chemical composition. The entire protein sample is utilized in crystallization experiments. Sparse-matrix screening and chemical gradient screening can be combined in one comprehensive 'hybrid' crystallization trial. The technology lends itself well to optimization by high-granularity gradient screening using optimization reagents such as precipitation agents, ligands or cryoprotectants.

Structural genomics of infectious disease drug targets: the SSGCID

“…With purified protein in hand, crystallization trials are set up in standard screens using two 96-condition sparse-matrix screens and two 96 grid-condition screens. In addition, a substantial number of SSGCID proteins have been screened using the Microcapillary Protein Crystallization System (MPCS) developed by the Protein Structure Initiative (PSI) ATCG3D technology center (Gerdts et al, 2008(Gerdts et al, , 2010. High-priority small-molecular-weight targets that fail to crystallize are selected for NMR-based analysis and structure determination at PNNL or UW-NMR (Tier 10).…”

Nanovolume optimization of protein crystal growth using the microcapillary protein crystallization system

“…Connection to the CrystalCard is achieved via the Teflon tubing and a polypropylene connector that forms an airtight seal to the port in the CrystalCard. The component liquids of the experiment are placed in the Teflon tubing of the syringe pumping system and delivered to the CrystalCard in a manner described previously using the MicroPlugger pumpcontrol software (Gerdts et al, 2008). The positioning of the fluid lines on the CrystalCard is noted in Fig.…”

“…The Accelerated Technologies Center for Gene to 3D Structures (ATCG3D) has developed the Microcapillary Protein Crystallization System (MPCS), which is a plug-based, microfluidic protein crystallization technology capable of quickly and easily setting up hundreds of batch-under-oil-style crystallization experiments (Gerdts et al, 2008). The MPCS is unique because it is capable of generating hundreds of nanovolume (10-20 nl) experiments, each containing a slightly different chemical composition (Gerdts et al, 2006;Zheng et al, 2003Zheng et al, , 2005.…”

Salvage and storage of infectious disease protein targets in the SSGCID high-throughput crystallization pathway using microfluidics

“…The plug-based microfluidic technology encompassed in the Plug Maker is able to thoroughly screen crystallization conditions by quickly and automatically generating concentration gradients for each condition that is screened. Details of the Plug Maker and its underlying technology have been published previously (Gerdts et al, 2008(Gerdts et al, , 2010Li et al, 2006;Zheng et al, 2003Zheng et al, , 2005. Using this technology, as many as 800 20 nl crystallization experiments can be set up in one CrystalCard (Emerald BioSystems; Fig.…”

“…2) using as little as 5 ml protein sample. Furthermore, although they only use up $10 nl of protein each, the experiments are large enough to produce diffraction-ready crystals (50-200 mm per side) that can be examined in situ or extracted from the peel-apart CrystalCard (Gerdts et al, 2008(Gerdts et al, , 2010Yadav et al, 2005).…”