Protein structure determination facility upgrade

蛋白质结构测定设施升级

• 批准号: RTI-2023-00404
• 负责人: Allingham, John
• 金额: $ 6.22万
• 依托单位: Queen's University
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=746078
• 关键词: Protein; structure; determination; facility; upgrade

Scope of research: All cellular functions are enabled by proteins. Understanding the structures of proteins and the architecture of protein complexes is the main gateway to understanding their functions. X-ray crystallography remains a gold standard method determining these macromolecular structures. Drs. Peter Davies and John Allingham lead research programs at Queen's University that have determined a broad spectrum of high-resolution protein structures using a home-source X-ray diffractometer that collects X-ray diffraction data from single proteins and protein complexes that have crystallized. Research programs to be supported: Dr. Davies is funded by NSERC to investigate the structures and mechanisms of proteins that mediate the interactions of bacteria with other cells or physical surfaces. Dr. Allingham is funded by NSERC to determine the structures and mechanisms of motor proteins that can change the structure of the actin and microtubule cytoskeletons, thereby affecting the movement and division of cells. Davies and Allingham also apply X-ray crystallography in drug discovery since ligands and compounds with high affinity can often be observed in the binding pockets of proteins. Need for the requested equipment: The X-ray crystallography laboratory at Queen's was constructed in 2008 and houses a Rigaku MicroMax007-HighFlux X-ray generator, an R-AXIS VI++ image-plate detector, and an X-Stream-2000 cryogenic cooling system. Normally, the X-Stream-2000 maintains crystal samples in a frozen, protected state during X-ray diffraction data collection by cooling them to -175 °C in an open flow nitrogen gas stream, but this machine is no longer functional nor serviceable. Without the ability to cool the protein crystals they are rapidly degraded by the X-ray beam. Thus, although the other components of the half million-dollar X-ray diffractometer are working well, we are completely unable to use it because of the broken cryostream. This application requests funding to purchase the Oxford Cryostream 1000 to replace this broken equipment. Training of HQP and sustainability: The X-ray crystallography laboratory was designed to allow experienced and novice crystallographers to complete a structural biology project from start to finish. Practical training and supervision in purifying target proteins, performing crystallization screening experiments, operating the diffractometer for crystal quality analysis and data collection, and solving protein structures is provided by Dr. Allingham, the X-ray lab director. Upgrades to this local research infrastructure is essential for these researchers to regain the ability to solve protein structures at Queen's and for maximizing their productivity during remote data diffraction data collections at synchrotron facilities. This equipment will also be critical for recruiting from the growing cohort of new scientists that are being trained in the fields of X-ray crystallography and cryo-electron microscopy.

研究范围：所有的细胞功能都是由蛋白质实现的。了解蛋白质的结构和蛋白质复合物的结构是了解其功能的主要途径。X射线晶体学仍然是确定这些大分子结构的金标准方法。彼得·戴维斯和约翰·阿林厄姆博士领导女王大学的研究项目，他们使用家庭来源的X射线衍射仪确定了高分辨率蛋白质结构的广谱，该X射线衍射仪收集了来自单一蛋白质和结晶蛋白质复合物的X射线衍射数据。戴维斯博士由NSERC资助，研究介导细菌与其他细胞或物理表面相互作用的蛋白质的结构和机制。Allingham博士由NSERC资助，以确定可以改变肌动蛋白和微管细胞骨架结构的运动蛋白的结构和机制，从而影响细胞的运动和分裂。Davies和Allingham还将X射线晶体学应用于药物发现，因为在蛋白质的结合口袋中经常可以观察到具有高亲和力的配体和化合物。对所需设备的需求：皇后学院的X射线晶体学实验室建于2008年，拥有理学MicroMax 007-HighFlux X射线发生器、R-AXIS VI++成像板探测器和X-Stream-2000低温冷却系统。通常情况下，X-Stream-2000在X射线衍射数据采集过程中，通过开放式氮气流将晶体样品冷却至-175 °C，从而使晶体样品保持在冷冻、受保护的状态，但这台机器已无法使用或维修。如果没有冷却蛋白质晶体的能力，它们会被X射线束迅速降解。因此，尽管这台价值50万美元的X射线衍射仪的其他部件工作正常，但由于低温流的损坏，我们完全无法使用它。该申请要求资助购买Oxford Cryostream 1000以更换此损坏的设备。 HQP和可持续性培训：X射线晶体学实验室旨在让经验丰富的和新手晶体学家从头到尾完成结构生物学项目。在纯化目标蛋白质，进行结晶筛选实验，操作衍射仪进行晶体质量分析和数据收集，并解决蛋白质结构的实际培训和监督是由博士阿林厄姆，X射线实验室主任。升级到这个本地的研究基础设施是必不可少的，这些研究人员重新获得解决蛋白质结构的能力在皇后，并最大限度地提高他们的生产力在远程数据衍射数据收集在同步加速器设施。这一设备对于招募越来越多的新科学家也至关重要，这些科学家正在接受X射线晶体学和低温电子显微镜领域的培训。