Temperature Control of Protein Crystal Nucleation
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TAL GROWTH
First Russian Crystallographic Congress
Temperature Control of Protein Crystal Nucleation V. V. Safronov*, N. V. Krivonogova, and V. I. Strelov Space Materials Science Laboratory, Shubnikov Institute of Crystallography, Federal Scientific Research Centre “Crystallography and Photonics,” Kaluga Branch, Russian Academy of Sciences, Kaluga, 248640 Russia *e-mail: [email protected] Received May 3, 2017
Abstract—A technique of temperature-controlled biocrystal nucleation, at which the growth onset of one crystal suppresses the development of other nuclei, is proposed. This is obtained by decreasing gradually the temperature at the point of expected growth at a rate sufficiently low to equalize the protein concentration (under conditions where material is captured by growing crystal) via diffusive and convective processes. An individual growth of lysozyme single crystals in a capillary is demonstrated. DOI: 10.1134/S1063774518020232
INTRODUCTION Growth of protein single crystals and other biological macromolecules is a necessary stage when determining their spatial structure by X-ray diffraction methods. The structural quality of these crystals is often insufficiently high (they are small or exhibit twinning), as a result of which high-resolution diffraction patterns cannot be recorded. Since protein crystals are generally formed via self-nucleation, one should consider the nonoptimality of initial growth conditions as one of the sources of these defects. In conventional methods, a supersaturation necessary for nucleation is formed either throughout the entire volume of crystallization sample (methods of vapor diffusion, bulk crystallization, and temperature shift [1, 2]) or in a sufficiently large part of the sample (diffusion
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Fig. 1. Schematic of crystallization experiment: (1) X-ray capillary with protein solution, (2) cold wedge, and (3) thermostated housing.
of precipitating agent through a gel [3]). A consequence is multiple nucleation, which often leads to growth of many small (and sometimes aggregated) microcrystallites. The number of nuclei can be reduced by providing local supersaturation (using the method of temperature-controlled crystallization in capillaries with a cold point [4]). The purpose of our study was to improve this method so as to make it possible to grow individual crystals by changing continuously temperature with time. EXPERIMENTAL Crystallization experiments were performed using a solution of hen egg lysozyme (BioChemica) in sodium acetate buffer (0.1 M, pH 4.6), with sodium chloride added as a precipitating agent. The protein and precipitating agent concentrations were, respectively, 30 and 25 mg/mL. The protein solution was centrifuged at a speed of 8000 rpm for 15 min. The solutions were filtered through Whatman cellulose filters with a pore size of 0.45 μm. Experiments were performed in a crystallization cell [5, 6] making it possible to set and maintain separately the temperatures of the crystallization chamber walls and the copper wedge supporting a horizo
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