Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip
| Autor: | Mario Mörl, Raphaël de Wijn, O. Hennig, Camille Noûs, Nicola Thome, Caroline Paulus, K. Rollet, Bernard Lorber, Claude Sauter, Vincent Olieric, Heike Betat |
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| Přispěvatelé: | Architecture et réactivité de l'ARN (ARN), Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS), Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Louis Pasteur - Strasbourg I |
| Rok vydání: | 2021 |
| Předmět: |
Enzyme substrate complex
Materials science Science & Technology biology General Immunology and Microbiology General Chemical Engineering General Neuroscience Microfluidics Active site Substrate (chemistry) Crystal growth Crystal structure General Biochemistry Genetics and Molecular Biology law.invention Enzymes Crystal Multidisciplinary Sciences Crystallography law biology.protein Science & Technology - Other Topics [SDV.BBM]Life Sciences [q-bio]/Biochemistry Molecular Biology Crystallization |
| Zdroj: | Journal of visualized experiments : JoVE Journal of visualized experiments : JoVE, JoVE, 2021, ⟨10.3791/61972⟩ |
| ISSN: | 1940-087X |
| DOI: | 10.3791/61972-v |
| Popis: | The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We describe a versatile microfluidic chip that enables the production of crystals by the efficient method of counter-diffusion. The convection-free environment provided by the microfluidic channels is ideal for crystal growth and useful to diffuse a substrate into the active site of the crystalline enzyme. Here we applied this approach to the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus in the presented example. After crystallization and substrate diffusion/soaking, the crystal structure of the enzyme:substrate complex was determined at room temperature by serial crystallography and the analysis of multiple crystals directly inside the chip. The whole procedure preserves the genuine diffraction properties of the samples because it requires no crystal handling. ispartof: JOVE-JOURNAL OF VISUALIZED EXPERIMENTS issue:169 ispartof: location:United States status: published |
| Databáze: | OpenAIRE |
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