High throughput sample delivery method for time resolved studies of enzyme reactions with X-ray and complementary techniques

高通量样品输送方法，用于利用 X 射线和补充技术进行酶反应的时间分辨研究

• 批准号: 10645032
• 负责人: Jan F Kern
• 金额: $ 61.41万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-16 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10645032
• 关键词: 3-Dimensional; Acoustics; Area; Back; Biochemical Reaction; Biological; Biological Models; Catalysis; Chemical Models; Chemicals; Collection; Communities; Consumption; Cryoelectron Microscopy; Crystallography; Data; Data Analyses; Data Collection; Deposition; Detection; Development; Dimensions; Dose; Drops; Drug Interactions; Drug Targeting; Electron Beam; Electrons; Ensure; Environment; Enzymatic Biochemistry; Enzymes; Feedback; Four-dimensional; Freezing; Funding; Future; Gases; Growth; In Situ; Individual; Injections; Kinetics; Lasers; Light; Liquid substance; Measurement; Measures; Metabolism; Methods; Microfluidics; Modeling; Modification; Molecular; Molecular Machines; Photons; Physiologic pulse; Physiological; Population; Process; Radiation induced damage; Reaction; Research; Resolution; Rest; Roentgen Rays; Sampling; Scheme; Science; Source; Stimulus; Structure; System; Techniques; Technology; Temperature; Testing; Therapeutic; Thermodynamics; Time; Transducers; Work; X ray diffraction analysis; biological systems; chemical property; computer studies; computerized tools; cryogenics; design; detection method; detector; electrical potential; enzyme model; enzyme substrate; experimental study; frontier; improved; instrumentation; interest; movie; multimodality; operation; pressure; prototype; simulation; structural biology; success; synchrotron radiation; temperature jump; tool; x-ray free-electron laser

Project Summary/Abstract One of the new frontiers in structural enzymology is the expansion from a three-dimensional to a truly four- dimensional approach by adding the time dimension to structural studies. While Synchrotron Radiation (SR) crystallography and cryo Electron Microscopy allow the determination of structures in minute detail they are in most cases performed on frozen static samples. With the advent of X-ray free electron lasers (XFELs) like the Linac Coherent Light Source (LCLS) at Stanford, and the development of the “probe before destroy” concept it now is possible to follow structural changes in enzymes in real time and under close to physiological conditions at room temperature (RT). Driven by the success of XFELs and recent advances in detector technology and storage ring and beam line design, several SR sources are also starting to offer time resolved crystallography at RT. These unprecedented capabilities will open new fields of research, not only in biomedical sciences but also in many other areas. Due to the “probe before destroy approach” utilized here, the samples generally need to be replaced after a single X-ray exposure. As biological samples of interest are often only available in scarce amounts, it is mandatory to develop a robust method to introduce the sample into the X-ray interaction region in a continuous manner that minimizes the required sample amount. In order to obtain true “molecular movies” of enzymes of biomedical importance in action, which will contribute to a deeper mechanistic understanding of these molecular machines, it is essential to synchronize the enzyme in the probed sample volume and initiate the reaction of interest in a temporally well-defined manner. Methods for reaction initiation can include mixing with a substrate/chemical compound, or utilize other stimuli such as light, temperature jump, or change in pH or electrical potential. In the frame of this proposal, we will continue the development of robust and versatile sample delivery and reaction triggering methods. We will also integrate multi-modal detection methods, combining X-ray diffraction with complementary in situ spectroscopic techniques to probe both global structures and chemical properties of enzymes concurrently. We will focus on the development of drop-on-demand methods based on acoustic transducer technology, but also explore other droplet dispensing technologies and microfluidics to substantially diminish/eliminate any sample wastage. We will improve the previously developed prototypes for depositing the drops on a moving support, such as a tape or wheel, that can circulate and is self-cleaning, for non-stop continuous operation at the XFEL or SR facility. Several methods for enzyme-substrate mixing will be tested, with emphasis on liquid-gas and liquid-liquid mixing, including with micron size droplet collision methods to achieve faster time resolution. Experiments on well-defined enzyme model systems will be accommodated by modeling approaches to design chemical mixing experiments and use feedback from measurements to optimize the design. These will be implemented at SR and XFEL beam lines and made available for the broader structural enzymology user community.

项目概要/摘要 结构酶学的新前沿之一是从三维扩展到真正的四维 同步辐射 (SR) 晶体学和冷冻电子显微镜可以确定结构的微小细节 随着 X 射线自由电子激光器 (XFEL) 的出现，大多数情况都是在冷冻静态样品上进行的。 斯坦福直线加速器相干光源 (LCLS)，以及“破坏前探测”概念的发展 现在可以在接近生理条件下实时跟踪酶的结构变化 在 XFEL 的成功和探测器技术的最新进展的推动下。 存储环和光束线设计，一些 SR 源也开始提供时间分辨晶体学 RT。这些前所未有的能力将开辟新的研究领域，不仅在生物医学领域，而且在生物医学领域。 在许多其他领域，由于此处使用的是“销毁前探测方法”，因此样本通常需要进行处理。 由于感兴趣的生物样本通常稀缺，因此在一次 X 射线照射后即可更换。 量，必须开发一种稳健的方法将样品引入 X 射线相互作用区域 以连续的方式最大限度地减少所需的样本量，以获得真实的“分子电影”。 在行动中具有生物医学重要性的酶，这将有助于更深入地理解 这些分子机器，必须同步探测样品体积中的酶并启动 以暂时明确的方式进行感兴趣的反应引发反应的方法可以包括混合。 使用底物/化合物，或利用其他刺激，例如光、温度跃变或 pH 值或 在此提案的框架内，我们将继续开发坚固且多功能的样品。 我们还将整合多模态检测方法，结合X射线。 衍射与互补的原位光谱技术来探测整体结构和化学 我们将同时关注酶的特性的开发。 声换能器技术，同时还探索其他液滴分配技术和微流体技术 大幅减少/消除任何样品浪费 我们将改进之前开发的原型。 将液滴沉积在可回收且具有自清洁功能的移动支撑物上，例如胶带或轮子， XFEL 或 SR 设施将采用多种酶-底物混合方法进行不间断连续操作。 经过测试，重点是液-气和液-液混合，包括微米尺寸的液滴碰撞方法 为了实现更快的时间分辨率，将在明确的酶模型系统上进行实验。 设计化学混合实验并使用测量反馈进行优化​​的建模方法 这些设计将在 SR 和 XFEL 光束线中实施，并可用于更广泛的结构。 酶学用户社区。

项目成果

Serial crystallography using automated drop dispensing.

使用自动液滴分配的连续晶体学。

• 发表时间: 2021-09-01
• 影响因子: 2.5
• 作者: Su, Zhen;Cantlon, Joshua;Douthit, Lacey;Wiedorn, Ma;Boutet, Sébastien;Kern, Jan;Yoon, Chun Hong;DePonte, Daniel
• 通讯作者: DePonte, Daniel

Solar energy conversion by photosystem II: principles and structures.

光系统 II 的太阳能转换：原理和结构。

• 发表时间: 2023-06
• 影响因子: 3.7
• 作者: Shevela, Dmitry;Kern, Jan F;Govindjee, Govindjee;Messinger, Johannes
• 通讯作者: Messinger, Johannes