Université de Strasbourg, Architecture et Réactivité de l'ARN, UPR 9002, CNRS, Institut de Biologie Moléculaire et Cellulaire; European XFEL GmbH.
Université de Strasbourg, Architecture et Réactivité de l'ARN, UPR 9002, CNRS, Institut de Biologie Moléculaire et Cellulaire; Biochemistry and Molecular Biology, Institute for Biochemistry, Leipzig University.
J Vis Exp. 2021 Mar 20(169). doi: 10.3791/61972.
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.
制备具有良好衍射性能的晶体以及在 X 射线分析之前对其进行处理，是生物晶体学研究的两个关键步骤。我们描述了一种通用的微流控芯片，该芯片可通过高效的逆流扩散方法生产晶体。微流道提供的无对流环境非常适合晶体生长，并可将基质扩散到结晶酶的活性部位。在本研究中，我们以嗜冷菌 Planococcus halocryophilus 的 CCA-添加酶为例应用了这种方法。结晶和基质扩散/浸泡后，通过连续结晶和在芯片内直接分析多个晶体，在室温下确定了酶：基质复合物的晶体结构。整个过程由于无需对晶体进行处理，因此保留了样品的真实衍射性能。
