Development of an Instant Freezing Sample Preparation System for Cryo-EM

项目摘要 项目名称：低温电子显微镜快速冷冻样品制备系统的研制 公司名称：dba蜂鸟精密机械有限公司 首席调查员：诺曼·萨蒙 摘要： 低温电子显微镜(Cryo-EM)领域作为一种 蛋白质结构表征技术。低温离子交换膜样品的制备 比其他成熟的技术如x射线结晶学更有优势，因为蛋白质不需要 被结晶以进行高分辨率的结构分析。而冷冻-EM样品的制备 技术使蛋白质保持其天然形式，低温EM在这两个质量上都遇到了瓶颈 和样品的吞吐量。该领域的最新进展集中在增加样本上 通过自动化制备技术提高产量。然而，更重要的问题是 低温电磁样品现在已经成为空气-水界面的蛋白质损伤。玻璃体前 冻结时，样品液滴中的蛋白质碰撞并粘在液滴表面，在那里它们 变性形成解开的单层。样品在冷冻前暴露的时间越长， 蛋白质与表面相互作用和变性的时间就越长。当前商业广告 系统不能直接解决与界面相关的问题，而是专注于 用薄而透明的冰自动生产样品。虽然这些改进是 对于减少冰层厚度的总体目标很有价值，它们忽略了对样品的损害 分子本身，防止用户重复获得高分辨率结构。 拟议的项目旨在通过以下方式革新低温EM的样品制备技术 探索一种产生更高质量的样品的方法，该样品表现出明显较小的损害 从空气-水界面。拟议的系统旨在限制样品暴露在空气-水中。 通过将冻结速度降低一个数量级来实现。为了减少损失 从具有高质量样品的空气-水界面，该系统的目标是在较少的时间内将样品玻璃化 超过10毫秒，有很大比例的冰适合高分辨率成像。这种“快速冻结” 系统还被构想为通过减少 冻结在网格上的过量水，形成更大面积的均匀冰和 每个网格的生物分子样本。蜂鸟科学旨在开发和测试背后的理论 系统处于第一阶段。

Project Summary Project Title: Development of an Instant Freezing Sample Preparation System for Cryo-EM Company Name: Hummingbird Precision Machine Co., dba Hummingbird Scientific Principal Investigator: Norman Salmon Summary: The field of cryogenic electron microscopy (cryo-EM) has rapidly been gaining traction as a structural characterization technique for proteins. Sample preparation for cryo-EM is advantageous over other proven techniques like x-ray crystallography as proteins do not need to be crystallized for high-resolution structural analysis. While the cryo-EM sample preparation technique keeps proteins in their native form, cryo-EM suffers from a bottleneck in both quality and throughput of samples. Recent advances in the field have focused on increasing sample throughput through automated preparation techniques. However, the more important problem in cryo-EM samples now has become protein damage from the air-water interface. Before vitreous freezing, proteins in the sample droplet collide and stick to the droplet surface, where they denature to form an unraveled monolayer. The more time the sample is exposed before freezing, the more time proteins have time to interact with the surface and denature. Current commercial systems fail to directly address the problems associated with the interface, focusing instead on the automated production of samples with thin, vitreous ice. While these improvements are valuable to the overall goals of reducing ice thickness, they ignore damage to the sample molecules themselves, preventing users from repeatably obtaining high-resolution structures. The proposed project aims to revolutionize sample preparation techniques for cryo-EM by exploring a method of generating higher quality samples that exhibit significantly less damage from the air-water interface. The proposed system aims to limit sample exposure to the air-water interface by reducing the rate of freezing by an order of magnitude. In order to reduce damage from the air-water interface with high quality samples, the system will aim to vitrify samples in less than 10 ms with a large percentage of ice suitable for high-resolution imaging. This “fast freezing” system also is also conceived to populate sample grids with consistently thin ice by reducing the amount of excess water that is frozen on the grid, creating larger areas of uniform ice and biomolecular sample per grid. Hummingbird Scientific aims to develop and test the theory behind the system in Phase I.