Crystallization Suite for the Structure Determination of Bacterial Cellulose Biosynthetic Proteins

这笔资金将用于购买晶体学套件，以支持申请人在细菌纤维素生物合成蛋白的结构-功能表征方面的研究项目。我们的功能研究与结构研究密切相关，因为这些领域相辅相成，可以详细了解酶活性和底物结合与关键结构褶皱和/或基序的关系。所需的晶体学套件将通过允许有效地生成和鉴定适合蛋白质结构测定的x射线衍射质量晶体来推进我们的结构研究。现代可编程孵化器将允许精确控制所需的最佳晶体生长参数。一个创新的立体显微镜配备了高倍率，一个紫外线模块和偏振光滤光片将大大提高我们的能力，明确区分蛋白质晶体从其他工件的视野。所要求的相机，软件和显微镜的自动化功能可以对图像清晰度，采集和处理进行特定控制，这对于可靠的文档记录和评估与再现性相关的晶体生长细节非常重要。低温储存杜瓦和晶体工具将有助于蛋白质晶体的适当低温保护，以尽量减少在运输和分析过程中对晶体的损害，同时也提供了一种长期储存的手段。因此，所要求的仪器将提供一个合适的结晶平台，提高我们生成适合蛋白质结构测定的x射线衍射质量晶体的能力。这些结果将提供对细菌纤维素生物合成的详细了解，并将导致发现与细菌种类的多糖合成和生物膜发育有关的重要原理。该结晶套件还将用于在威尔弗里德劳里埃大学创建一个晶体学资源中心，从而确保结构-功能生物化学领域的学员具备先进的技能/技术，使他们在进入加拿大劳动力市场或追求高等教育时具有很强的竞争力。

This grant will purchase a crystallography suite to support the applicant's research program in the structure-function characterization of bacterial cellulose biosynthetic proteins. Our functional studies are closely intertwined with our structural research as these areas complement each other to provide a detailed understanding of how enzyme activity and substrate binding relates to critical structural folds and/or motifs. The requested crystallography suite will advance our structural research by permitting the efficient generation and identification of X-ray diffraction quality crystals suitable for protein structure determination. Modern programmable incubators will allow for precise control over parameters needed for optimal crystal growth. An innovative stereomicroscope equipped with high magnification, a UV-light module and polarized light filters will greatly enhance our ability to unambiguously distinguish protein crystals from other artifacts in the field of view. The requested camera, software and automated features for the microscope enable specific control over image clarity, acquisition and processing which are important for reliable documentation and assessing crystal growth details related to reproducibility. Cryogenic storage dewars and crystal tools will facilitate the proper cryoprotection of protein crystals in order to minimize damage to crystals during transport and analysis while also providing a means for long-term storage. Thus, the requested instruments will provide a proper crystallization platform that enhances our ability to generate X-ray diffraction quality crystals suitable for protein structure determination. These results will provide a detailed understanding of the biosynthesis of bacterial cellulose and will lead to the discovery of important principles relating to polysaccharide synthesis and biofilm development across bacterial species. This crystallization suite will also serve to create a crystallography resource centre at Wilfrid Laurier University, thereby ensuring that trainees in the field of structure-function biochemistry are equipped with advanced skills/techniques that make them highly competitive upon entering Canada's workforce or pursuing higher education.