Hyperquenching Cryocooler for Biomolecular X-ray Crystallography

项目摘要 该项目的总体目标是设计、开发和商业化紧凑型低温冷却仪器， 快速、可重复和可靠地冷冻保存生物分子晶体，以用于X射线冷冻组织化学照相术。x射线 晶体学是确定蛋白质分子结构的最有力和最广泛使用的工具， 病毒、核酸和生物分子复合物。这些结构对现代分子生物学至关重要 以及用于病症和疾病的药物治疗的发展。插入冷却晶体 液氮和在T=100 K时收集数据简化了存储、运输和处理， 增加了可以从每个晶体获得的X射线数据量。然而，冷却过程本身 会损坏晶体。包括病毒、膜蛋白和生物分子复合物在内的许多重要靶标 可能很难成功冷却。此外，目前的冷却方法，这是耗时的， 并且手工完成，导致冷却结果和衍射质量的大变化，并且可能不准确， 捕捉室温/生物温度分子结构的所有重要细节。拟议的文书将 以用户可调节的速率提供可靠的半自动冷却，从而提供最大的控制， 设计和实施冷却方案的灵活性。冷却速率高达80，000 K/s，比原来大100倍 比目前的最佳做法，将消除结晶冰形成在冷却过程中， 需要冷冻保护剂浓度。入门级，高通量和研究仪器将 使用模块化平台开发，以及快速响应薄膜温度传感器， 测量这些仪器将提供的大冷却速率。研究晶体的性质和冷却 在确定X射线衍射结果中相互作用的协议将验证这些仪器的实用性， 为最有效地使用它们制定规程。这些工具将大大提高效率， 生物分子晶体学管道的有效性，并为学术，政府和 工业科学家致力于推进我们对生命和疾病的分子机制的理解。

Project Abstract The overall goal of this project is to design, develop and commercialize compact cryocooling instruments for fast, reproducible and reliable cryopreservation of biomolecular crystals for X-ray cryocrystallography. X-ray crystallography is the most powerful and widely used tool for determining the molecular structures of proteins, viruses, nucleic acids and biomolecular complexes. These structures are critical to modern molecular biology and to the development of pharmaceutical treatments for conditions and diseases. Plunge cooling crystals in liquid nitrogen and collecting data at T=100 K simplifies storage, transport and handling, and dramatically increases the amount of X-ray data that can be obtained from each crystal. However, the cooling process itself damages crystals. Many important targets including viruses, membrane proteins and biomolecular complexes can be very difficult to successfully cool. Moreover, current cooling methodologies, which are time-consuming and done by hand, lead to large variability in cooling outcomes and diffraction quality, and may not accurately capture all salient details of the room/biological temperature molecular structure. The proposed instruments will provide reliable, semi-automated cooling at user adjustable rates, and so provide maximum control and flexibility in designing and implementing cooling protocols. Cooling rates of up to 80,000 K/s, 100 times larger than in current best practice, will eliminate crystalline ice formation during cooling and dramatically reduce required cryoprotectant concentrations. Entry-level, high-throughput and research instruments will be developed using a modular platform, as well as fast-response thin-film temperature sensors needed to measure the large cooling rates these instruments will deliver. Studies of how crystal properties and cooling protocols interact in determining X-ray diffraction outcomes will validate the utility of these instruments and establish protocol for their most effective use. These instruments will significantly improve the efficiency and efficacy of biomolecular crystallography pipelines, and provide new capabilities to academic, government and industrial scientists working to advance our understanding of the molecular mechanisms of life and disease.