Laboratory of Biomolecular Structure and Function

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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.