A Single Entity Method for Controlled Nucleation and Crystal Growth

控制成核和晶体生长的单一实体方法

• 批准号: 10720470
• 负责人: Gangli Wang
• 金额: $ 36.45万
• 依托单位: GEORGIA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10720470
• 关键词: 3-Dimensional; Adopted; Area; Binding; Biological Process; Biomedical Research; Cell Nucleus; Chemicals; Complex; Crystal Formation; Crystallization; Data Collection; Development; Diffusion; Disadvantaged; Disease; Drug Design; Electron Microscopy; Embryo; Feedback; Formulation; Future; Goals; Growth; Habits; Hydrogen; Individual; Inherited; Insulin; Ions; Kinetics; Lipids; Liquid substance; Location; Magic; Measurement; Membrane Proteins; Methodology; Methods; Monitor; Muramidase; Names; Nature; Needles; Neutrons; Noise; Nucleic Acids; Optical Methods; Optics; Pharmacologic Substance; Phase; Phase Transition; Physiological; Play; Preparation; Process; Program Development; Proteins; Protons; Quantitative Evaluations; R-factor; Reproducibility; Research; Resolution; Roentgen Rays; Role; Sampling; Shapes; Signal Transduction; Site; Sodium Chloride; Solvents; Source; Structure; Surface; System; Techniques; Temperature; Thermodynamics; Thinness; Time; Transport Process; Uncertainty; Variant; X-Ray Crystallography; active control; biomacromolecule; design; drug development; improved; insight; macromolecule; migration; nano; nanodevice; nanoscale; outcome prediction; predictive signature; programs; prototype; real time monitoring; success; technology development; tool; two-dimensional; vapor

The structures of biomacromolecules at atomic resolution (< 2.0-2.5 Å) are of enormous importance to understand their physiological functions and roles in diseases. An exemplary critical need of high atomic resolution is to resolve the location of proton/hydrogen which plays vital roles in various biological processes. Deuteration renders neutron scattering techniques unique advantages in high contrast (signal/background) to locate D/H. Like X-ray crystallography which has contributed majority of known biomolecule structures, high quality single crystals are the prerequisites for both X-ray and neutron data collection. It is worth mentioning that despite the recent progresses in electron microscopy techniques, true atomic resolution remains a formidable challenge to achieve. Lower resolution structures are associated with ambiguity and could mislead basic biomedical research as well as drug design/development applications. With the understanding on the fundamental limitations and technical hurdles associated with currently adopted ensemble-based methods, we propose to develop a single-entity method (named NanoAC) which will offer unprecedented capability to synthesize crystals one at a time, under real-time monitoring and with predictive crystal quality. A single nanotip will be employed to spatially confine supersaturation as the sole nucleation site. Electroanalytical and optical methods will monitor the whole crystallization process in real-time to capture quantitative signatures for the nucleation and crystal growth at single entity resolutions. Those signatures will enable active controls in kinetic transitions, and be quantitatively correlated with its diffraction quality and/or crystal habits. The insights will inform crystal synthesis such that nucleation kinetics and growth rates of each individual crystal will be finetune to improve crystal quality and to tune crystal size/habits. Prototype soluble proteins, nucleic acids and membrane proteins will be used as defined in this early-stage technology development program. The new toolbox, once established, will provide paradigm-shift capabilities to improve the crystal quality in diffraction and size/habit controls, to tackle challenging material systems currently not-crystallizable, and also feature high efficiency in time and/or materials. The overarching goal will be pursued through three interrelated aims. Aim 1 will establish real-time monitoring signatures for the generalization of NanoAC to crystallize soluble biomacromolecules and complexes. Aim 2 will correlate diffraction quality and crystal habits with monitoring signatures. Aim 3 will further develop single nanopipettes as ‘magic wand’ to crystallize membrane proteins.

生物大分子在原子分辨率（< 2.0-2.5 μ m）下的结构对于 了解它们的生理功能和在疾病中的作用。一个示范性的关键需要高原子 分辨是分辨在各种生物过程中起重要作用的质子/氢的位置。 氘化使得中子散射技术在高对比度（信号/背景）方面具有独特的优势， 找到D/H像X射线晶体学贡献了大多数已知的生物分子结构一样， 高质量的单晶是X射线和中子数据收集的先决条件。值得一提的是 尽管电子显微镜技术最近取得了进展，但真正的原子分辨率仍然是一个令人生畏的问题。 挑战实现。较低分辨率的结构与模糊性有关，可能会误导基本的 生物医学研究以及药物设计/开发应用。在达成谅解后， 与目前采用的基于集成的方法相关的基本限制和技术障碍，我们 建议开发一种单实体方法（命名为NanoAC），该方法将提供前所未有的能力， 在实时监控下并以可预测的晶体质量一次合成一个晶体。单个纳米尖端 将用于在空间上限制过饱和作为唯一的成核位点。电分析和光学 方法将实时监测整个结晶过程，以捕获 成核和晶体生长在单一实体的分辨率。这些特征将使动力学中的主动控制成为可能 转变，并与其衍射质量和/或晶体习性定量相关。这些见解将为 晶体合成，使得每个单独晶体的成核动力学和生长速率将被微调， 改善晶体质量和调整晶体尺寸/习性。原型可溶性蛋白质、核酸和膜 蛋白质将按照该早期技术开发计划中的定义使用。新工具箱，一旦 建立，将提供范式转移能力，以改善晶体质量的衍射和大小/习惯 控制，以解决目前不可结晶的具有挑战性的材料系统，并具有高效率， 时间和材料。将通过三个相互关联的目标来实现总体目标。目标1将建立 实时监测NanoAC的推广特征，使可溶性生物大分子结晶， 配合物目标2将把衍射质量和晶体习性与监控特征联系起来。目标3将进一步 开发单纳米移液管作为“魔杖”来结晶膜蛋白。