Abstract
A simple technique for high-throughput protein crystallization in ionically cross-linked polysaccharide gel beads has been developed for contactless handling of crystals in X-ray crystallography. The method is designed to reduce mechanical damage to crystals caused by physical contact between crystal and mount tool and by osmotic shock during various manipulations including cryoprotection, heavy-atom derivatization, ligand soaking, and diffraction experiments. For this study, protein crystallization in alginate and κ-carrageenan gel beads was performed using six test proteins, demonstrating that proteins could be successfully crystallized in gel beads. Two complete diffraction data sets from lysozyme and ID70067 protein crystals in gel beads were collected at 100 K without removing the crystals; the results showed that the crystals had low mosaicities. In addition, crystallization of glucose isomerase was carried out in alginate gel beads in the presence of synthetic zeolite molecular sieves (MS), a hetero-epitaxic nucleant; the results demonstrated that MS can reduce excess nucleation of this protein in beads. To demonstrate heavy-atom derivatization, lysozyme crystals were successfully derivatized with K2PtBr6 within alginate gel beads. These results suggest that gel beads prevent serious damage to protein crystals during such experiments.
Highlights
The methods and technologies for crystallization of proteins for use in X-ray diffraction experiments have advanced dramatically in recent years
In the conventional crystal manipulation process, manual handling with the commercial crystal mounting loops such as Hampton CryoLoop (Hampton Research), MiTeGen MicroLoop (MiTeGen LLC), and LithoLoop (Molecular Dimensions Inc.) often causes mechanical damage to the protein crystals due to physical contact between crystal and loop, resulting in the deterioration of diffraction quality
Protein crystallization in gel-filled capillary tubes [6,9], gel slices [10], rehydratable gel [11], and hydrogel beads [12] have been reported, and such methods have been successfully used to crystallize proteins. These techniques can potentially prevent mechanical damage caused by physical contact with the crystals
Summary
The methods and technologies for crystallization of proteins for use in X-ray diffraction experiments have advanced dramatically in recent years. Protein crystallization in gels such as agarose, agar, and silica gels is a well-known technique for production of high-quality crystals [1,2,3]; in such methods, crystal quality is improved due to reductions in convective flow, crystal sedimentation, nucleation, and twinning [2,4,5]. Protein crystallization in gel-filled capillary tubes [6,9], gel slices [10], rehydratable gel [11], and hydrogel beads [12] have been reported, and such methods have been successfully used to crystallize proteins These techniques can potentially prevent mechanical damage caused by physical contact with the crystals. Ionically cross-linked alginatecalcium gel bead technique may allow high-throughput crystallization screening and X-ray diffraction studies in which crystals grown in gel beads are exposed to X-rays without being first removed from the beads, permitting acquisition of diffraction data through contactless handling of crystals
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