Cryo-EM grid preparation using gas dynamic virtual nozzles

使用气体动态虚拟喷嘴进行冷冻电镜网格制备

• 批准号: 10009848
• 负责人: Osman Bilsel
• 金额: $ 16.81万
• 依托单位: MICROGRADIENT FLUIDICS LLC
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2021-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10009848
• 关键词: 3-Dimensional; Address; Air; Algorithms; Apoferritin; Area; Automation; Biological; Carbon; Complex; Coupled; Cryoelectron Microscopy; Crystallization; Deposition; Development; Devices; Diffusion; Ethane; Feedback; Freezing; Gases; Heterogeneity; Ice; Immersion; Liquid substance; Macromolecular Complexes; Microfluidic Microchips; Microfluidics; Molecular Weight; Monitor; Motor; Preparation; Proteins; Research Personnel; Resolution; Sampling; Shapes; Space Perception; Speed; Spottings; Stream; Structure; Surface; System; Technology; Thick; Thinness; Time; Tracer; Translating; Translations; Visualization; Water; aqueous; arm; base; carbon fiber; detector; fluorescence imaging; instrument; instrumentation; macromolecule; millisecond; particle; protein complex; prototype; reconstruction; structural biology; tool; virtual

Project Summary Recent advances in cryo-electron microscopy (cryoEM), such as the development of direct detectors, automation and 3D particle reconstruction algorithms, have transformed structural biology by enabling investigators to obtain near atomic resolution structures without the need to grow crystals. A significant challenge that limits the wider applicability of cryoEM as a structural tool is the preparation of suitable samples in thin (< 100 nm) layers of vitreous ice in the approximately micron-sized holes of a substrate grid. This arises from the large surface-area-to-volume ratio of the thin aqueous layers prior to freezing of the thin liquid layer in a cryogen. Biological macromolecules preferentially localize to the air-water interface in the thin liquid layer, giving rise to preferred orientations or, in some cases, denaturation. A significant development in overcoming this problem has been the development of droplet based approaches to depositing samples on the grids. This minimizes the dwell time of the protein in the water layer, i.e., the time between spotting of the sample on the grid and plunge- freezing of the liquid layer. The spot-to-plunge time of state-of-the-art instrumentation (~10 ms), however, is still nearly three orders of magnitude longer than the time for diffusion of sample to the air-water interface (~ 10-100 microseconds). The proposed instrument will use thin liquid jets formed using gas dynamic virtual nozzles (GDVN) together with an ultrarapid (~50 m/s) plunging system to reduce the spot-to-plunge time to the microsecond regime. By minimizing this time to less than the diffusion time to the air-water interface, preferential orientation and degradation issues can be minimized. Additionally, the device has the potential to precisely control the ice layer thickness and readily adapt to time-resolved studies. Successful development has the potential to significantly widen the biological macromolecules and scientific questions that can be addressed by high-resolution structure determination using single particle cryoEM.

项目摘要 低温电子显微镜（cryoEM）的最新进展，例如 直接探测器，自动化和3D粒子重建算法， 通过使研究人员能够获得近原子分辨率， 结构，而无需生长晶体。一个重大挑战，限制了更广泛的 cryoEM作为一种结构工具的适用性是在薄层中制备合适的样品， （< 100 nm）层的玻璃状冰在基板的大约微米大小的孔中 网格这是由于薄水层的表面积与体积之比很大 然后在冷冻剂中冷冻薄液体层。生物大分子 优先定位于薄液体层中的空气-水界面，引起 优选的取向，或者在某些情况下，变性。中的一项重大发展 克服这个问题的是基于液滴的方法的发展 将样品沉积在网格上。这最小化了蛋白质在膜中的停留时间。 水层，即，在网格上点样和插入之间的时间- 液体层的冻结。最先进仪器的点到切入时间 然而，（~10 ms）仍然比时间长近三个数量级 样品扩散到空气-水界面（~ 10-100微秒）。拟议 仪器将使用气体动力学虚拟喷嘴（GDVN）形成的薄液体射流 与超快速（~50 m/s）插入系统一起使用，以缩短点到插入时间 到微秒级。通过将该时间最小化到小于扩散时间， 空气-水界面、优先取向和降解问题可以最小化。 此外，该装置具有精确控制冰层厚度的潜力， 很容易适应时间分辨的研究。成功的开发有可能 大大拓宽了生物大分子和科学问题， 通过使用单粒子cryoEM的高分辨率结构测定来解决。