Microcrystal preparation for serial femtosecond X-ray crystallography of bacterial copper amine oxidase
Microcrystal preparation for serial femtosecond X-ray crystallography of bacterial copper amine oxidase
复制标题
细菌铜胺氧化酶连续飞秒X射线晶体学微晶制备
DOI：
10.1107/s2053230x21008967
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Okajima Toshihide
中科院分区：
文献类型：
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作者：
Murakawa Takeshi;Suzuki Mamoru;Arima Toshi;Sugahara Michihiro;Tanaka Tomoyuki;Tanaka Rie;Iwata So;Nango Eriko;Tono Kensuke;Hayashi Hideyuki;Fukui Kenji;Yano Takato;Tanizawa Katsuyuki;Okajima Toshihide
使用X射线自由电子激光的连续飞秒X射线晶体学（SFX）的最新进展为在非冻结条件下确定无辐射损伤的蛋白质结构铺平了道路。然而，大规模制备尺寸均匀的高质量微晶是SFX的先决条件，这一直是其广泛应用的障碍。在这里，一个方便的方法制备高品质的细菌醌蛋白酶，铜胺氧化酶的球形节杆菌，微晶的报道。该方法包括机械粉碎大晶体（5-15 mm 3），将粉碎的晶体接种到酶溶液中，并在26°C的环境温度下静置1 h，导致快速形成3-5 µm均匀尺寸的微晶。 在SPring-8埃紧凑型自由电子激光器设施的SFX测量中，微晶衍射X射线的分辨率超过2.0μ m。在2.2 μ m分辨率下确定的无损伤结构与先前使用同步加速器X射线辐射通过低温晶体学确定的结构基本相同。
Recent advances in serial femtosecond X-ray crystallography (SFX) using X-ray free-electron lasers have paved the way for determining radiation-damage-free protein structures under nonfreezing conditions. However, the large-scale preparation of high-quality microcrystals of uniform size is a prerequisite for SFX, and this has been a barrier to its widespread application. Here, a convenient method for preparing high-quality microcrystals of a bacterial quinoprotein enzyme, copper amine oxidase from Arthrobacter globiformis, is reported. The method consists of the mechanical crushing of large crystals (5–15 mm3), seeding the crushed crystals into the enzyme solution and standing for 1 h at an ambient temperature of ∼26°C, leading to the rapid formation of microcrystals with a uniform size of 3–5 µm. The microcrystals diffracted X-rays to a resolution beyond 2.0 Å in SFX measurements at the SPring-8 Angstrom Compact Free Electron Laser facility. The damage-free structure determined at 2.2 Å resolution was essentially identical to that determined previously by cryogenic crystallography using synchrotron X-ray radiation.