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Anaerobic fixed-target serial crystallography

DOI: 10.1107/S2052252520010374 DOI Help

Authors: Patrick Rabe (University of Oxford) , John Beale (Diamond Light Source) , Agata Butryn (Diamond Light Source) , Pierre Aller (Diamond Light Source) , Anna Dirr (University of Oxford) , Pauline A. Lang (University of Oxford) , Danny N. Axford (Diamond Light Source) , Stephen Carr (Research Complex at Harwell) , Thomas M. Leissing (University of Oxford) , Michael A. Mcdonough (University of Oxford) , Bradley Davy (Diamond Light Source) , Ali Ebrahim (Diamond Light Source; University of Essex) , Julien Orlans (Diamond Light Source; University of Lyon) , Selina L. S. Storm (Diamond Light Source) , Allen M. Orville (Diamond Light Source) , Christopher J. Schofield (University of Oxford) , Robin L. Owen (Diamond Light Source)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Iucrj , VOL 7

State: Published (Approved)
Published: September 2020
Diamond Proposal Number(s): 19458

Open Access Open Access

Abstract: Cryogenic X-ray diffraction is a powerful tool for crystallographic studies on enzymes including oxygenases and oxidases. Amongst the benefits that cryo-conditions (usually employing a nitro­gen cryo-stream at 100 K) enable, is data collection of di­oxy­gen-sensitive samples. Although not strictly anaerobic, at low temperatures the vitreous ice conditions severely restrict O2 diffusion into and/or through the protein crystal. Cryo-conditions limit chemical reactivity, including reactions that require significant conformational changes. By contrast, data collection at room temperature imposes fewer restrictions on diffusion and reactivity; room-temperature serial methods are thus becoming common at synchrotrons and XFELs. However, maintaining an anaerobic environment for di­oxy­gen-dependent enzymes has not been explored for serial room-temperature data collection at synchrotron light sources. This work describes a methodology that employs an adaptation of the `sheet-on-sheet' sample mount, which is suitable for the low-dose room-temperature data collection of anaerobic samples at synchrotron light sources. The method is characterized by easy sample preparation in an anaerobic glovebox, gentle handling of crystals, low sample consumption and preservation of a localized anaerobic environment over the timescale of the experiment (<5 min). The utility of the method is highlighted by studies with three X-ray-radiation-sensitive Fe(II)-containing model enzymes: the 2-oxoglutarate-dependent L-arginine hy­droxy­lase VioC and the DNA repair enzyme AlkB, as well as the oxidase isopenicillin N synthase (IPNS), which is involved in the biosynthesis of all penicillin and cephalosporin antibiotics.

Journal Keywords: anaerobic crystallization; oxygen-employing enzymes; penicillin biosynthesis; 2-oxoglutarate/α-ketoglutarate oxygenases; serial crystallography

Subject Areas: Technique Development, Biology and Bio-materials, Chemistry

Instruments: I24-Microfocus Macromolecular Crystallography