New X-ray Diffractometer and Detector for Purdue Macromolecular Crystallography

总结 通过X射线晶体学以原子分辨率测定大分子结构可以说是 上个世纪生命科学最伟大的技术成就。通过揭示分子 这项技术极大地加深了我们对健康和疾病的理解 并加速了更具选择性、更有效和更安全的药物的开发。蛋白质的故事 晶体学在普渡大学开始于1964年与招聘迈克尔罗斯曼，在该领域的先驱。一 正式的生物物理学培训计划是在1989年与NRSA机构博士前培训补助金， 与此同时，马基基金会（Markey Foundation）授予了数百万美元的赠款，为招聘提供资金 此外，我们还需要增加现有X射线设施中的教员和X射线发生器。装备精良 和专家教师致力于推进分子生物物理学和初级科学家的培训，普渡大学 产生了许多成功的研究者，包括吴昊（哈佛）、梁彤（哥伦比亚）、马文·哈克特（Marvin Hackert (UT奥斯汀）和杰克约翰逊（斯克里普斯）。约翰·特斯默，这个提议的PI，也是普渡大学的实习生。 普渡大学也是该领域创新的重要枢纽。例如，Rossmann博士和Dr. 珍妮特·史密斯分别是分子置换和MAD定相技术的关键贡献者， 这是目前世界范围内最常用的两种定相技术。普渡建筑的新时代 生物学开始了。2009年，该大学开设了霍克迈耶结构生物学大厅，一个3000万美元的设施， 和高分子晶体学核心的新家。普渡大学重申了其对结构性 通过2019年购买RockImager Duo水晶酒店和成像系统来更新核心， 和牛津低温系统800系列液氮冷却器我们的T32分子生物物理学培训的重新启动 计划（资助2019-24）现在每年支持7名学员（Tesmer PI）。这项建议的具体目的是 通过更换我们老化的~30岁的X射线发生器来满足关键需求，这些发生器无法再修复， 一个现代化的，可靠的，具有成本效益的MicroMax-007高频旋转阳极发生器耦合到同步加速器 质量，Dectris Eiger 2混合光子计数探测器。发电机及其先进的光学系统将提供一个 在晶体上提供明亮的90 µm小X射线束，优化小样品的信噪比。大155 x163 毫米探测器将是弱反射的理想选择，75微米的小像素可以分辨密集的反射。A 4- Circle Kappa测角仪将增加数据收集的灵活性。新的设置将提高X射线通量>100倍， 探测器的灵敏度大约是成像板的10倍，从而提高了我们的整体性能。 性能提高了大约三个数量级，并能够从衍射数据中收集上级衍射数据。 即使是最具挑战性的水晶新的配置将大大有利于科学和教育 至少20名教师的使命，不仅提供高分辨率的生物学见解，他们的目标，而且 加速开发针对癌症和神经疾病的新疗法。

Summary Macromolecular structure determination at atomic resolution via X-ray crystallography is arguably one of the greatest technical achievements in the life sciences over the last century. By shedding light on molecular mechanisms essential for life, the technique has dramatically deepened our understanding of health and disease and accelerated the development of drugs that are more selective, potent, and safe. The story of protein crystallography at Purdue began in 1964 with the recruitment of Michael Rossmann, a pioneer in the field. A formal biophysics training program was instituted in 1989 with an NRSA Institutional Predoctoral Training Grant, coinciding with the award of a multi-million dollar Markey Foundation grant, which provided funds for hiring additional faculty and the X-ray generators currently in the existing X-ray facility. Armed with excellent facilities and expert faculty dedicated to advancing molecular biophysics and the training of junior scientists, Purdue produced many successful investigators including Hao Wu (Harvard), Liang Tong (Columbia), Marvin Hackert (UT Austin), and Jack Johnson (Scripps). John Tesmer, the PI of this proposal, is likewise a Purdue trainee. Purdue has also served as an important hub for innovation in the field. For example, Dr. Rossmann and Dr. Janet Smith were key contributors to the techniques of molecular replacement and MAD phasing, respectively, which are now the two most commonly used phasing techniques world-wide. A new era in Purdue structural biology has begun. In 2009, the university opened Hockmeyer Hall of Structural Biology, a 30 million dollar facility and the new home of the Macromolecular Crystallography core. Purdue reaffirmed its commitment to structural biology by updating the core with the 2019 purchase of a RockImager Duo crystal hotel and imaging system, and an Oxford Cryosystems 800 Series liquid nitrogen cooler. A reboot of our T32 Molecular Biophysics Training Program (funded 2019-24) now supports 7 trainees per year (Tesmer PI). The specific aim of this proposal is to fulfill a critical need by replacing our aging ~30 year old X-ray generators, which can no longer be repaired, with a modern, reliable, and cost effective MicroMax-007 HF rotating anode generator coupled to a synchrotron quality, Dectris Eiger2 hybrid photon counting detector. The generator and its advanced optics will provide a brilliant, small 90 µm X-ray beam at the crystal, optimizing signal-to-noise for small samples. The large 155x163 mm detector will be ideal for weak reflections, with small 75 µm pixels to resolve closely spaced reflections. A 4- circle kappa goniometer will add flexibility in data collection. The new setup will improve X-ray flux >100-fold, and the detector sensitivity is about 10 times greater than imaging plates, thereby improving our overall performance by about three orders of magnitude and enabling the collection of superior diffraction data from even the most challenging crystals. The new configuration will greatly benefit the scientific and educational mission of at least 20 faculty by not only providing high resolution biological insights into their targets, but also accelerating the development of novel therapeutics targeting cancer and neurological disease.