Hyperquenching Cryocooler for Biomolecular X-ray Crystallography

项目摘要 该项目的总体目标是设计、开发紧凑型低温冷却设备并将其商业化 用于X射线晶体学的生物分子晶体快速、可重复、可靠的冷冻保存。X射线 结晶学是确定蛋白质分子结构的最强大和最广泛使用的工具， 病毒、核酸和生物分子复合体。这些结构对现代分子生物学至关重要。 以及开发治疗疾病和疾病的药物疗法。将冷却晶体放入 液氮和在T=100K下收集数据简化了存储、运输和处理，并极大地 增加可从每个晶体获得的X射线数据量。然而，冷却过程本身 破坏水晶。许多重要的靶标包括病毒、膜蛋白和生物分子复合体 可以很难成功降温。此外，目前的冷却方法非常耗时 和手工操作，导致冷却结果和衍射质量变化很大，并且可能不准确 捕捉室温/生物温度分子结构的所有显著细节。拟议的文书将 以用户可调的速度提供可靠的半自动冷却，从而提供最大限度的控制和 灵活设计和实施冷却协议。冷却速度高达8万K/S，100倍 与当前的最佳实践相比，将在冷却过程中消除结晶冰的形成并显著减少 所需的冷冻保护剂浓度。入门级、高通量和研究仪器将是 使用模块化平台开发，以及需要快速响应的薄膜温度传感器 测量这些仪器将提供的大冷却速度。晶体性质和冷却方式的研究 在确定X射线衍射结果时相互作用的协议将验证这些仪器的实用性，并 为最有效地利用它们制定议定书。这些仪器将显著提高效率和 生物分子晶体管道的功效，并为学术、政府和 工业科学家致力于促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.