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Microcrystal preparation for serial femtosecond X-ray crystallography of bacterial copper amine oxidase.


ABSTRACT: 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.

SUBMITTER: Murakawa T 

PROVIDER: S-EPMC8488853 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature

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Microcrystal preparation for serial femtosecond X-ray crystallography of bacterial copper amine oxidase.

Murakawa Takeshi T   Suzuki Mamoru M   Arima Toshi T   Sugahara Michihiro M   Tanaka Tomoyuki T   Tanaka Rie R   Iwata So S   Nango Eriko E   Tono Kensuke K   Hayashi Hideyuki H   Fukui Kenji K   Yano Takato T   Tanizawa Katsuyuki K   Okajima Toshihide T  

Acta crystallographica. Section F, Structural biology communications 20210921 Pt 10


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 fr  ...[more]

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