Department of Molecular and Structural Biochemistry, North Carolina State University; Neutron Scattering Division, Oak Ridge National Laboratory.
Department of Molecular and Structural Biochemistry, North Carolina State University; Neutron Scattering Division, Oak Ridge National Laboratory;
J Vis Exp. 2020 Dec 1(166). doi: 10.3791/61903.
Neutron crystallography is a structural technique that allows determination of hydrogen atom positions within biological macromolecules, yielding mechanistically important information about protonation and hydration states while not inducing radiation damage. X-ray diffraction, in contrast, provides only limited information on the position of light atoms and the X-ray beam rapidly induces radiation damage of photosensitive cofactors and metal centers. Presented here is the workflow employed for the IMAGINE and MaNDi beamlines at Oak Ridge National Laboratory (ORNL) to obtain a neutron diffraction structure once a protein crystal of suitable size (> 0.1 mm) has been grown. We demonstrate mounting of hydrogenated protein crystals in quartz capillaries for neutron diffraction data collection. Also presented is the vapor exchange process of the mounted crystals with D2O-containing buffer to ensure replacement of hydrogen atoms at exchangeable sites with deuterium. The incorporation of deuterium reduces the background arising from the incoherent scattering of hydrogen atoms and prevents density cancellation caused by their negative coherent scattering length. Sample alignment and room temperature data collection strategies are illustrated using quasi-Laue data collection at IMAGINE at the High Flux Isotope Reactor (HFIR). Furthermore, crystal mounting and rapid freezing in liquid nitrogen for cryo-data collection to trap labile reaction intermediates is demonstrated at the MaNDi time-of-flight instrument at the Spallation Neutron Source (SNS). Preparation of the model coordinate and diffraction data files and visualization of the neutron scattering length density (SLD) maps will also be addressed. Structure refinement against neutron data-only or against joint X-ray/neutron data to obtain an all-atom structure of the protein of interest will finally be discussed. The process of determining a neutron structure will be demonstrated using crystals of the lytic polysaccharide monooxygenase Neurospora crassa LPMO9D, a copper-containing metalloprotein involved in the degradation of recalcitrant polysaccharides via oxidative cleavage of the glycosidic bond.
中子晶体学是一种结构技术，可用于确定生物大分子中氢原子的位置，从而提供关于质子化和水合状态的重要机械信息，而不会引起辐射损伤。相比之下，X 射线衍射只能提供关于轻原子位置的有限信息，并且 X 射线束会迅速引起光敏辅助因子和金属中心的辐射损伤。本文介绍了在橡树岭国家实验室 (ORNL) 的 IMAGINE 和 MaNDi 光束线获得中子衍射结构的工作流程，前提是已经生长出具有合适尺寸 (>0.1 毫米) 的蛋白质晶体。我们演示了将氢化蛋白质晶体装入石英毛细管中进行中子衍射数据收集的过程。还介绍了装有晶体的蒸汽交换过程，用含有 D2O 的缓冲液交换可交换位置的氢原子，以确保用氘取代。氘的掺入减少了来自氢原子非相干散射的背景，并防止了由于其负的相干散射长度而导致的密度抵消。使用准劳埃数据在 HFIR 的 IMAGINE 上进行了样品对准和室温数据收集策略的说明。此外，还演示了在散裂中子源 (SNS) 的 MaNDi 飞行时间仪器上进行晶体安装和快速液氮冷冻以捕获不稳定的反应中间体的方法。模型坐标和衍射数据文件的准备以及中子散射长度密度 (SLD) 图的可视化也将得到解决。最后将讨论针对仅中子数据或针对联合 X 射线/中子数据进行结构精修以获得感兴趣的蛋白质的全原子结构。使用纤维二糖单加氧酶 Neurospora crassa LPMO9D 的晶体来演示确定中子结构的过程，该酶是一种含铜的金属蛋白，通过糖苷键的氧化裂解参与难降解多糖的降解。
