Cryo-EM grid preparation using gas dynamic virtual nozzles

项目摘要 低温电子显微镜的最新进展，如电子显微镜的发展 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接近原子的分辨率来改变结构生物学 无需生长晶体的结构。一项重大挑战，限制了更广泛的 低温EM作为一种结构工具的适用性是在薄层中制备合适的样品 在衬底的大约微米大小的孔中形成(&lt；100 nm)层玻璃冰 栅格。这是由于薄水层的表面积与体积之比大所致。 在冷冻制冷剂中的薄液层之前。生物大分子 优先定位于薄液层中的空气-水界面，产生 优先取向，或在某些情况下，变性。在…方面的重大发展 克服这一问题的是基于液滴的方法的发展 把样品放在栅格上。这最大限度地减少了蛋白质在 水层，即从样品在网格上发现到下沉之间的时间- 冻结液体层。最先进的仪器仪表从点到潜的时间 然而，(~10ms)仍然比以下时间长近三个数量级 样品扩散到空气-水界面(约10-100微秒)。建议数 仪器将使用由气动虚拟喷嘴(GDVN)形成的稀薄液体射流 配合超高速(~50米/S)潜水系统，缩短点到潜水时间 到微秒制度。通过将此时间最小化到小于扩散到 可以最大限度地减少空气-水界面、择优取向和降解问题。 此外，该装置有可能精确控制冰层厚度和 很容易适应时间分辨的研究。成功的开发具有以下潜力 大大拓宽了生物大分子和科学问题的范围 通过使用单粒子低温电子显微镜的高分辨率结构确定来解决。

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.