Anaerobic fixed-target serial crystallography
| Autor: | Thomas M. Leissing, Pauline A. Lang, Michael A. McDonough, Bradley Davy, Robin L. Owen, Stephen B. Carr, Patrick Rabe, Julien Orlans, Pierre Aller, Anna Dirr, Allen M. Orville, Selina L. S. Storm, Christopher J. Schofield, A. Ebrahim, Danny Axford, John H. Beale, A. Butryn |
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| Přispěvatelé: | DIAMOND Light source, Université Grenoble Alpes - École supérieure du professorat et de l'éducation - Académie de Grenoble (UGA ESPE Grenoble), Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), ISIS Neutron and Muon Source (ISIS), STFC Rutherford Appleton Laboratory (RAL), Science and Technology Facilities Council (STFC)-Science and Technology Facilities Council (STFC), Biologie Fonctionnelle, Insectes et Interactions (BF2I), Institut National des Sciences Appliquées de Lyon (INSA Lyon), Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Wellcome Trust102593210734/Z/18/ZRoyal Society of LondonRSWF\R2\182017Deutsche Akademie fur Naturforscher Leopoldina, Germany Biotechnology and Biological Sciences Research Council (BBSRC)102593Royal Society Wolfson Fellowship RSWF\R2\182017Cancer Research UK Wellcome Investigator Award in Science 210734/Z/18/Z, Laboratoire de Modélisation et Simulation Multi Echelle (MSME), Centre National de la Recherche Scientifique (CNRS)-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12)-Université Paris-Est Marne-la-Vallée (UPEM) |
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
inorganic chemicals
penicillin biosynthesis AlkB Isopenicillin N synthase 010402 general chemistry 01 natural sciences Biochemistry law.invention 03 medical and health sciences law [CHIM]Chemical Sciences General Materials Science Sample preparation Reactivity (chemistry) serial crystallography lcsh:Science 030304 developmental biology chemistry.chemical_classification 0303 health sciences biology oxygen-employing enzymes General Chemistry Condensed Matter Physics Research Papers Synchrotron 0104 chemical sciences Crystallography Enzyme 2-oxoglutarate/alpha-ketoglutarate oxygenases chemistry biology.protein anaerobic crystallization lcsh:Q Protein crystallization Anaerobic exercise 2-oxoglutarate/α-ketoglutarate oxygenases |
| Zdroj: | IUCrJ International Union of Crystallography journal International Union of Crystallography journal, International Union of Crystallography 2020, 7 (5), pp.901-912. ⟨10.1107/S2052252520010374⟩ IUCrJ, Vol 7, Iss 5, Pp 901-912 (2020) |
| ISSN: | 2052-2525 |
| DOI: | 10.1107/S2052252520010374⟩ |
| Popis: | An effective and simple-to-implement approach for anaerobic room-temperature data collection is described and demonstrated by application to dioxygen utilizing enzymes. 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 ( |
| Databáze: | OpenAIRE |
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