In situX-ray analysis of protein crystals in low-birefringent and X-ray transmissive plastic microchannels

Abstract

Plastic microchannel crystallization template designs made from inexpensive cyclic olefin copolymers have been shown to be low-birefringent, X-ray transmissive and compatible with microfluidic fabrication in restricted geometry. The model proteins thaumatin, lysozyme and bacteriorhodopsin demonstrated the feasibility of conducting counter-diffusion equilibration within the new plastic configuration. Crystals of each of these proteins were directly evaluated in situ using synchrotron radiation and their diffraction quality was evaluated without invasive manipulation or cryofreezing. Protein crystals able to produce complete X-ray data sets were used to calculate electron-density maps for structure determination. Fluidic crystallization in the plastic platform was also coupled with a commercialized automated imager and an in situ X-ray scanner that allowed optical and X-ray inspection of crystallization hits. The results demonstrate the feasibility of rapid nanovolume counter-diffusion crystallization experiments without the need for additional instrumentation.

Clustering procedures for the optimal selection of data sets from multiple crystals in macromolecular crystallography

“…(ii) small crystals that diffract very weakly on standard beamlines can instead produce respectable diffraction patterns using intense microbeams; and (iii) small-sized crystals can produce weak but sufficiently interpretable diffraction patterns when they are exposed directly inside crystallization plates (Watanabe et al, 2002;Ng et al, 2008;le Maire et al, 2011;Soliman et al, 2011;. The obvious advantage of this in situ technique is that the whole time-consuming procedures behind loop fishing and crystal cooling can be avoided, thus making the method fast and amenable to automation.…”

X-CHIP: An Integrated Platform for High-Throughput Protein Crystallography

“…Several devices have been developed, and even commercialized (Topaz® crystallizer, Fluidigm Corp., CA, USA; The Crystal Former, Microlytic Inc., MA, USA) that utilize free interface diffusion (FID) (Hansen et al, 2002). Other chips employ nanochannels to create counter-diffusion crystallization (Hasegawa et al, 2007, Ng et al, 2008, Dhouib et al, 2009 or nanodroplets that simulate batch crystallization (Zheng et al, 2003). There are two clear, parallel implications in all these devices.…”

“…There are two clear, parallel implications in all these devices. They are all striving to increase efficiency of the hit identification process, and are offering the possibility of in situ X-ray analysis and, in favorable cases, diffraction data collection for structure determination (Zheng et al, 2004, Hansen et al, 2006, Ng et al, 2008, May et al, 2008, Dhouib et al, 2009). …”

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

“…New technologies to improve protein crystallization success rates are the focus of continuous research and technology development (Fox et al, 2008;Ng, Clark et al, 2008;Li et al, 2009Li et al, , 2010Hansen et al, 2002;Cherezov et al, 2008Cherezov et al, , 2009Dhouib et al, 2009;Sauter et al, 2007;Hansen & Quake, 2003). Protein crystals are often so difficult to produce that crystallographers are willing to try a new crystallization technology, even if it might provide only a small chance at crystallization success.…”