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

Abstract

The Microcapillary Protein Crystallization System (MPCS) is a microfluidic, plug-based crystallization technology that generates X-ray diffraction-ready protein crystals in nanolitre volumes. In this study, 28 out of 29 (93%) proteins crystallized by traditional vapor diffusion experiments were successfully crystallized by chemical gradient optimization experiments using the MPCS technology. In total, 90 out of 120 (75%) protein/precipitant combinations leading to initial crystal hits from vapor diffusion experiments were successfully crystallized using MPCS technology. Many of the resulting crystals produced high-quality X-ray diffraction data, and six novel protein structures that were derived from crystals harvested from MPCS CrystalCards are reported.

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).…”

Microfluidics: From crystallization to serial time-resolved crystallography

“…In particular, COC has seen widespread adoption as the polymer film of choice for X-ray compatible devices, 46 including simple channel structures for counterdiffusion, 56,58,60,61,126 droplet-based devices, 108,109,118 and larger-scale X-ray compatible wellplates. [127][128][129][130][131][132][133][134][135][136][137][138][139][140] However, further decreasing the device thickness to achieve the signal-to-noise levels required for microcrystallography is a significant materials' challenge.…”

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).…”