Hyperquenching Cryocooler for Biomolecular X-ray Crystallography

用于生物分子 X 射线晶体学的超淬火制冷机

• 批准号: 8719826
• 负责人: Robert Newman
• 金额: $ 56.48万
• 依托单位: MITEGEN, LLC
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8719826
• 关键词: Affect; Biological; Biological Process; Biomedical Research; Complex; Controlled Study; Cryopreservation; Crystallography; Data; Development; Disease; Electronics; Ethane; Excision; Film; Gases; Genes; Germ Cells; Goals; Government; Hand; Housing; Human; Humidity; Ice; Lead; Letters; Life; Liquid substance; Market Research; Marketing; Measurement; Measures; Membrane Proteins; Methodology; Methods; Molecular; Molecular Biology; Molecular Medicine; Molecular Structure; Nitrogen; Nucleic Acids; Outcome; Performance; Pharmaceutical Preparations; Pharmacologic Substance; Pharmacology; Phase; Preparation; Price; Process; Propane; Property; Proteins; Protocols documentation; Public Health; Research; Resolution; Resources; Roentgen Rays; Safety; Sampling; Scientist; Small Business Innovation Research Grant; Source; Speed; Stem cells; Structure; Students; Surface; Synchrotrons; Technology; Temperature; Testing; Time; Training; Viral Proteins; Virus; Work; X ray diffraction analysis; X-Ray Crystallography; X-Ray Diffraction; beamline; cold temperature; commercialization; cryogenics; design; egg; flexibility; improved; instrument; prevent; prototype; public health relevance; response; sensor; sperm cell; tool

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.

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 用于 X 射线冷冻晶体学的生物分子晶体的快速、可重复且可靠的冷冻保存。 X射线 晶体学是确定蛋白质分子结构最强大且使用最广泛的工具， 病毒、核酸和生物分子复合物。这些结构对于现代分子生物学至关重要 以及开发针对病症和疾病的药物治疗方法。将冷却晶体放入 液氮并在 T=100 K 时收集数据简化了存储、运输和处理，并显着 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 损坏晶体。许多重要靶标，包括病毒、膜蛋白和生物分子复合物 成功冷却可能非常困难。此外，当前的冷却方法非常耗时 手工完成，会导致冷却结果和衍射质量存在很大差异，并且可能不准确 捕捉室温/生物温度分子结构的所有显着细节。拟议文书将 以用户可调的速率提供可靠的半自动冷却，从而提供最大程度的控制和 设计和实施冷却协议的灵活性。冷却速率高达 80,000 K/s，提高 100 倍 比目前的最佳实践，将消除冷却过程中结晶冰的形成，并大大减少 所需的冷冻保护剂浓度。入门级、高通量和研究仪器将 使用模块化平台以及快速响应薄膜温度传感器开发 测量这些仪器将提供的大冷却速率。研究晶体特性和冷却方式 协议在确定 X 射线衍射结果时相互作用将验证这些仪器的实用性 建立最有效使用的协议。这些仪器将显着提高效率和 生物分子晶体学管道的功效，并为学术界、政府和 工业科学家致力于增进我们对生命和疾病分子机制的理解。