Laboratory of Biomolecular Structure and Function

项目摘要 （生物分子结构与功能实验室） OUHSC X射线核心设施于1998年启动，由NIH IDEA授予Paul Weigel博士， 生物化学和分子生物学系。该设施最近更名为 生物分子结构与功能（LBSF）LBSF提供专业知识和评估工具， COBRE的个人项目涉及晶体学和小角X射线的各种关键技术 散射研究（SAXS）的蛋白质和核酸。晶体学技术包括筛选 晶体在室温下的衍射质量，晶体冷冻保护剂筛选，晶体冷冻保护 高压下，衍射数据采集，衍射数据处理，空间群归属，结构 结构测定、结构精修、结构分析、结构解释和图形制备。的 SAXS相关的方法包括动态光散射检查聚集体和测量多分散性 然后将样品送往国家设施进行小角X射线散射数据收集。在COBRE的第一阶段， 升级了理学MicroMax 007 X射线发生器，板Wyatt板读取动态灯 用于高通量研究的散射仪器和用于制作晶体图像的Leica显微镜。的 一种新的X射线发生器能产生四倍强度的更小直径的X射线束。这些功能至关重要 用于筛选COBRE研究项目中常见的小型弱衍射晶体。的 俄克拉荷马州医学研究基金会捐赠了一台Mar345成像板X射线检测器，一台Varimax HF光学器件 用于将惰性气体引入到蛋白质腔中的系统、Oxford低温系统和MSC Xe-Siter装置 用于相位实验理学X射线系统通过增加第二个工作台进行了修改， 启用将Mar345系统添加到X射线发生器的第二个端口。RaxisIV系统是 带有屏蔽罩，使用户能够在收集数据时将晶体安装在Mar系统上 在Rigaku系统上。此设置使两个组能够同时收集数据，或者一个组能够 同时收集两个晶体的衍射数据。新的X射线发生器已被用于筛选 蛋白质和RNA晶体的衍射质量和发展冷冻条件。读板DLS 已经使用装置来筛选促进蛋白质储存的蛋白质稳定性的缓冲液， 结晶试验DLS也已用于筛选SAXS样品的聚集体的存在之前， 运往国家设施的SAXS光束线。在第一阶段，10组参与了SAXS研究。期间 第二阶段，将增加一个综合分子建模单元，以促进密集的计算工作， 作为候选药物分子的病毒筛选、分子动力学模拟、分子置换 与phenix-rosetta，和SAXS数据与结构的ensenbles分子建模。该单位将 由来自OMRF的Tim Mather博士每天管理，他也是OUHSC的附属成员 生物化学与分子生物学系。此外，一个艺术罗宾斯结晶机器人为 将在LBSF中加入在水立方相中的完整膜蛋白的结晶， 在OUHSC校园进行膜蛋白的晶体学研究。MCL中的结晶机器人 OU-Norman校区将继续用于可溶性蛋白质。MCL还将用于高- 使用Plate-Mate和Pilatus探测器对晶体进行批量筛选。OUHSC一直在为以下人员提供工资 Simon Terzyan博士于2015年受聘管理LBSF的日常运营。 他维护仪器，安排用户，并提供晶体学培训。他还安装了 大多数新工具。2015年，LBSF成为由 研究副总裁（VPR）。此套件的管理核心负责开具发票并管理 LBSF的独立账户。VPR为LBSF设立了监督委员会。该委员会是 由COBRE副主任主持。LBSF的主任Blaine Mooers博士负责这项工作。 委员会作为一个临时成员，促进LBSF在研讨会上，监督实验室经理，写道 设备拨款申请，并为VPR，EAC和NIH撰写进度报告。他本是 自2008年起担任该设施的负责人。Mooers博士和Terzyan博士在以下领域拥有超过45年的综合经验： 大分子晶体学，这将有助于项目研究人员在他们的建议取得成功 第二阶段的结构研究。

PROJECT SUMMARY (Laboratory for Biomolecular Structure and Function) The OUHSC X-ray Core Facility was initiated in 1998 with a NIH IDeA grant to Dr. Paul Weigel, former chair of the Department of Biochemistry and Molecular Biology. The facility was recently renamed the Laboratory of Biomolecular Structure and Function (LBSF). The LBSF provides expertise and assess to instruments to the individual COBRE projects on a variety of techniques critical to crystallographic and small angle X-ray scattering studies (SAXS) of proteins and nucleic acids. The crystallographic techniques include screening crystals for diffraction quality at room temperature, crystal cryo-protectant screening, crystal cryprotection under high pressure, diffraction data collection, diffraction data processing, space group assignment, structure determination, structure refinement, structure analysis, structure interpretatoin, and figure preparation. The SAXS-related methods include dynamic light scattering to check for aggregates and to measure polydispersity prior to sending the samples for SAXS data colleciton at national facilities. In Phase I of the COBRE, the facility was upgraded with a new Rigaku MicroMax 007 X-ray generator, a plate Wyatt plate-reading dynamic light scattering instrument for high throughput studies, and a Leica microscope for making images of crystals. The new X-ray generator gives a four-fold more intense X-ray beam of smaller diameter. These features are critical for the screening the small, weakly diffracting crystals common in the COBRE research projects. The Oklahoma Medical Research Foundation donated a Mar345 image plate X-ray dectector, a Varimax HF optic system, an Oxford cryosystem, and a MSC Xe-Siter apparatus for introducing noble gases into protien cavities in crystals for phasing experiments. The Rigaku X-ray system was modifed by adding a second bench to enable the addition of the Mar345 system to the second port of the X-ray generator. The RaxisIV system was enclosed with shielding that enables users to mount crystals on the Mar system while data are being collected on the Rigaku system. This setup has enabled two groups to collect data simultaneously or one group to collect diffraction data from two crystals at the same time. The new X-ray generator has been used to screen protein and RNA crystals for diffraction quality and to develop cryo-conditions. The plate-reading DLS apparatus has been used to screen buffers for those that promote protein stability for protein storage and crystallization trials. DLS has also been used to screen SAXS samples for the presence of aggregates prior to shipment to SAXS beam lines at national facilities. In Phase I, ten groups participated in SAXS studies. During Phase II, an Integrative Molecular Modeling Unit will be added to faciltate intenstive computational work such as virutal screening for candidate drug molecules, molecular dynamics simulations, molecular replacement with phenix-rosetta, and molecular modeling of SAXS data with ensenbles of structures. This unit will be managed on a daily basis by Dr. Tim Mather from OMRF who is also an adjunct member of the OUHSC Department of Biochemistry and Molecualr Biology. In addtion, an Art Robbins crystallization robot for the crystallizaton of integral membrane proteins in the lipidic cubic phase will be added to the LBSF to facilitate crystallograpic studies of membrane proteins on the OUHSC campus. The crystallization robots in the MCL on the OU-Norman campus will continue to be used for soluble proteins. The MCL will also be used for high- troughput screening of crystals with the Plate-Mate and Pilatus detector. OUHSC has been providing salary for a staff sceintist, Dr. Simon Terzyan, who was hired in 2015 to manage the day-to-day operatons of the LBSF. He maintains the instruments, schedules users, and provides training in crystallography. He has also installed most of the new instruments. In 2015, the LBSF became a core lab in the suite of core labs supported by the Vice President of Research (VPR). The administrative core of this suite issues invoices and manages the independent account of the LBSF. The VPR setup an oversight committee for the LBSF. This committee is chaired by the COBRE associate director. The Director of the LBSF, Dr. Blaine Mooers, serves on this committee as an ad hoc member, promotes the LBSF in seminars, oversees the Lab Manager, writes equipment grant applications, and writes progress reports for the VPR, EAC, and NIH. He had been the director of this facility since 2008. Drs Mooers and Terzyan have over 45 years of combined experience in macromolecular crystallography that will help the Project Investigators achieve success in their proposed structural studies during Phase II.