Protein structure determination facility upgrade

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。Drs。彼得·戴维斯和约翰·阿林厄姆在女王大学领导了一个研究项目，他们利用家用x射线衍射仪确定了高分辨率蛋白质结构的广谱图，该衍射仪收集了单个蛋白质和结晶的蛋白质复合物的x射线衍射数据。研究项目支持：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。阿林厄姆博士是由NSERC资助，以确定运动蛋白的结构和机制，可以改变肌动蛋白和微管细胞骨架的结构，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，因为高亲和力的配体和化合物通常可以在蛋白质的结合口袋中观察到。所需设备：女王大学的x射线晶体学实验室于2008年建成，包括一台Rigaku MicroMax007-HighFlux x射线发生器，一台R-AXIS VI++图像板探测器和一台X-Stream-2000低温冷却系统。通常情况下，X-Stream-2000在x射线衍射数据收集过程中通过在开放流氮气流中将晶体样品冷却到-175°C来保持晶体样品处于冷冻保护状态，但这台机器不再功能或可用。由于无法冷却蛋白质晶体，它们被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良好，但由于冷冻气流的破坏，我们完全无法使用它。本申请申请资金购买牛津低温流1000来更换这个损坏的设备。HQP和可持续性培训：x射线晶体学实验室旨在让有经验的和新手晶体学家从头到尾完成一个结构生物学项目。x射线实验室主任阿林厄姆博士提供了纯化靶蛋白、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以及解决蛋白质结构的实践培训和监督。升级当地的研究基础设施对于这些研究人员重新获得在皇后大学解决蛋白质结构的能力以及在同步加速器设施中最大限度地提高他们在远程数据衍射数据收集中的生产力至关重要。该设备对于招募正在接受x射线晶体学和低温电子显微镜领域培训的新科学家也至关重要。

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.