A convenient tool for gas derivatization using fine-needle capillary mounting for protein crystals

Nobuhiro Mizuno,Takashi Kumasaka,Masatomo Makino

doi:10.1107/s0909049513021584

Nobuhiro Mizuno, Takashi Kumasaka + Show 1 more

Open Access

https://doi.org/10.1107/s0909049513021584

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Abstract

Gas derivatization of protein crystals is useful not only to analyse gas-binding proteins but also to solve the phase problem of X-ray crystallography by using noble gases. However, the gas pressurization tools for these experiments are often elaborate and need to release the gas before flash-cooling. To simplify this step, a procedure using a fine-needle capillary to mount and flash-cool protein crystals under the pressurization of gases has been developed. After the crystals are picked up with the capillary, the capillary is sealed with an adhesive and then connected directly to a gas regulator. The quality of the diffraction data using this method is comparable with that of data from conventional pressurization procedures. The preparation of xenon-derivatives of hen egg-white lysozyme using this method was a success. In the derivatives, two new xenon binding sites were found and one of their sites vanished by releasing the gas. This observation shows the availability of flash-cooling under gas pressurization. This procedure is simple and useful for preparing gas-derivative crystals.

Highlights

Gas adsorption to proteins is an interesting phenomenon as a function of gas-binding proteins and in the determination of the crystal structures of proteins using noble gases
Gas-pressurized cryogenic samples have been prepared using a capillary (Schiltz et al, 1997), an original pressure cell (Soltis et al, 1997; Hayakawa et al, 2008) or some commercially available tools: XCell (Oxford Cryosystems, Oxford, UK), Cryo-Xe-Siter (Rigaku, The Woodlands, TX, USA) and Xenon Chamber (Hampton Research, Aliso Viejo, CA, USA), which can be used for the xenon pressurization up to several MPa
The lysozyme crystals in the fine-needle capillary were successfully flash-cooled by a N2 cryostream under the pressurization of xenon gas

Summary

Introduction

Gas adsorption to proteins is an interesting phenomenon as a function of gas-binding proteins and in the determination of the crystal structures of proteins using noble gases. Pressurization with the noble gases can be used to introduce a heavy atom into the structure. This method has been shown to be effective for structural determinations (Stowell et al, 1996; Prangeet al., 1998). Despite these developments, only about a dozen studies have determined protein structures by noble gases, because the gas pressurization procedure is complicated to handle

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