PROJECT SUMMARY/ ABSTRACT The X-ray diffraction system, in the laboratory of Prof. Joshua Sakon (PI X-ray crystallography core), has played and will continue to play a key role in the statewide research efforts in the structure determination of biomolecules. His involvement in the Arkansas INBRE began in 2001 when the Arkansas BRIN was first funded through a grant from the National Center for Research Resources. Dr. Sakon co-directs the Biotechnology Core and in that role helps Arkansas INBRE network faculty and students access University of Arkansas core facilities. This budget is for the acquisition of the HyPix-6000HE Hybrid Photon Counting Detector and Universal Kappa 4-Axis Goniometer for the X-Ray diffraction system. The instrumentation will enhance user access to new technology while replacing outdated equipment that is no longer serviceable by the manufacturer. With the new installation, the deinstallation of Saturn-92 and AFC9, Beryllium window of left port will also be required. The system, which was purchased in 2003, has provided an excellent return on investment for 17 years. Eighty-one structures solved in the facility are validated and made accessible from the Protein Data Bank (PDB). The facility provided 78.4% of three-dimensional structures deposited from our state of Arkansas. During the period 2003−2021, the coordinates were frequently downloaded (1,685,267 times) and examined using the website tools implemented by PDB (503,322 times) and led our state in impact (87.9% of downloads and views). These structures are aiding in understanding protein architectures and designing new molecules. The detector/goniometer portion of the system is in desperate need of upgrade. However, the old detector is no longer supported by the manufacturer Replacing the existing detector/goniometer portion of the system will extend the life of the X-ray diffraction system as well as increase the functionality and usability of the system. The new generation X-ray detector will produce higher quality data and the software that now comes with the detector/goniometer replacement will leverage advanced new algorithms developed in recent years and will be capable of extracting more accurate information even with the same signal to noise ratio. The investment will extend the capacity of the instrument from being a system dedicated to protein crystallography to one that is capable of also generating high-quality analytics for small molecules. With the proposed upgrade of the system, for less money than a new small molecule crystallography system, the facility can continue to serve the needs of INBRE-, COBRE-, and as well as other NIH-supported investigators on this campus and across the state.

Acquisition of a new goniometer and detector for the X-Ray diffraction system The X-ray diffraction system, in the laboratory of Prof. Joshua Sakon (PI X-Ray Crystallography Core), has played and will continue to play a key role in the statewide research efforts in the structure determination of biomolecules. His involvement in the Arkansas INBRE began in 2001 when the Arkansas BRIN was first funded through a grant from the National Center for Research Resources. Dr. Sakon co-directs the Arkansas INBRE Biotechnology Core and in that role helps network faculty and students access University of Arkansas core facilities. The system was purchased in 2003 and has provided an excellent return on investment for 17 years. The detector/goniometer portion of the system is in desperate need of upgrade. The 17-year-old detector is no longer supported by the manufacturer as the manufacturer has moved away from CCD (charge-coupled device) to a new technology with much improved resolution. Replacing the existing detector/goniometer portion of the system will extend the life of the X-ray diffraction system as well as increase the functionality and usability of the system. The new generation of X-ray detector will produce higher quality data than what can be done with the existing X-ray detector and the software that now comes with the detector/goniometer. The replacement leverages advanced new algorithms developed in the recent decade and will enable the extraction of more accurate information with better confidence even with the same signal to noise ratio. The investment will extend the capacity of the instrument from being a system dedicated to protein crystallography to one that is capable of also generating high-quality small molecule results, which will help the core facility to stay at the cutting edge of biomedical research, as well as provide needed service to additional research labs in the state. Research Impact: Funds are being sought to update the existing Rigaku rotating anode generator X-ray diffraction system that is used in macromolecular crystallographic research. When purchased in 2003, the system came with a CCD detector that represented cutting edge technology at that time. In recent years, a new detector technology has replaced CCD. This new technology, HPAD (Hybrid Pixel Array Detector) a.k.a. HPC (Hybrid Photon Counting) detector, was developed at the CERN Super Collider to measure X-rays with very low noise and high dynamic range. Detectors based on HPAD now dominate macromolecular beamlines at synchrotrons around the world and Rigaku’s implementation of this technology (HyPix family of detectors) has now made it the technology of choice for academic laboratories. There are many benefits of HPAD technology. For macromolecular crystallography, the extremely low noise characteristic means that a researcher can measure weak samples more accurately because there is no noise buildup in long exposures. The upgrade will benefit researchers like Prof. Mohammad Halim of University of Arkansas-Fort Smith who is characterizing the interaction of inhibitors and main protease of SARS-CoV-2 (letter attached). UAFS is a primarily undergraduate institution in the Arkansas INBRE network. The interaction of the lead compounds with the drug target obtained through crystallography may allow the next cycle of chemical optimization. Crystallography remains better suited to yield precise atomic coordinates of macromolecules under a few hundred kilodaltons in size, while the ability to probe larger, potentially more disordered assemblies is a distinct advantage over cryo-EM. The system which was purchased in 2003 has provided an excellent return on investment for 17 years. Eighty- one structures solved in the facility are validated and made accessible from the Protein Data Bank (PDB). The facility provided 78.4% of 3D structures deposited from Arkansas. The coordinates were downloaded or examined using PDB online tools $300,000 time per year. The facility led our state in impact (87.9% of downloads and views). These structures are aiding in understanding protein architectures and designing new molecules. NIH, DOE, and NSF grants of 14 different investigators used the instrument. Three investigators are from primarily undergraduate institutions (PUI) in the Arkansas INBRE network and one PI is from University of Arkansas at Pine Bluff, an HBCU and PUI in the network. Crystal structures obtained by X-Ray Crystallography Core paved the way for translational research. Prof. Sakon’s curiosity in crystallography and enzymology has resulted in 15 patents, several of which have been company licensed. He was elected as a National Academy of Inventors Fellow in 2020. His involvement in industry has led him to be a founder of two companies, BiologicsMD LLC and S3 Biologics LLC. The diffractometer in the X-ray core uses Cu X-ray radiation. In the past, this X-ray source would limit the diffractometer to macromolecular crystallography since small molecule crystallography generally relies on Mo X- ray radiation. Recently a new paradigm has arisen as a result of new detector and software technology. Many small molecule crystallographers in fact prefer Cu X-ray radiation because of the ability to measure smaller samples and collect data faster. The proposed upgrade will allow the system to be used for macromolecular crystallography as well as small molecule crystallography because the new goniometer and the software that comes with it allow both types of experiments to be performed. The current software is only valid for macromolecular crystallography. The University of Arkansas is one of the few nationally-ranked chemistry departments that does not currently have small molecule capabilities. With the proposed upgrade, this problem will be rectified and for less money than a new small molecule crystallography system would cost. Inorganic and organometallic catalysts studied by Prof. Stefan Kilyanek’s lab at UA has biomedical implications (Letter attached). The fundamental electrochemical reactions of molybdenum oxygen that are being studied in his lab have implications for metalloenzymes. His lab and other labs are sending samples via FedEx to other universities around the country for this essential data. The upgrade proposed in this application will extend the capacity of the instrument from being a system dedicated to protein crystallography to one that is capable of also generating high-quality small molecule results. Prof. Qinglong Jiang of University of Arkansas-Pine Bluff (PUI & HSBC) needs crystal structures of light emitting crystals (Letter attached). Crystallography can provide high-resolution dynamic information as a function of time, temperature and pressure. User fees and vouchers will ensure equipment maintenance. Continued support for a successful macromolecular crystallography research program as well as the addition of small molecule crystallography capabilities to investigators at UA and PUIs across the state at a price less than a new small molecule system is good investment for the university and the Arkansas INBRE. Educational Impact: While the upgrades of the goniometer and detector are critical to not only serve the expanding needs of our on-campus users, but also investigators at our the PUIs within the state. The upgrades will also provide a new multi-core Windows 10 control PC to run the advanced algorithms and a software package, CrysAlisPro. A multi-site/multi-user license come with the purchase, thus the CrysAlisPro software can be given to every user on the UA campus as well as those at Arkansas INBRE partners PUIs. All students, postdocs, and research advisors can have access to the software on their individual computers to process and catalogue their data. This will allow for many datasets to be processed off-site, thus freeing the control PC for additional data collection. The software can also be used to process data from equipment of other vendors as well as synchrotron sources such as the Advanced Light Source in Berkeley, California and the Advanced Photon Source in near Chicago. It also affords students the opportunity to learn the fundamentals of X-ray diffraction using a software package that allows them to easily determine structural models and how to resolve some non- trivial issues with crystal structure determinations that may occur with some samples. CrysAlisPro can process diffraction data from high pressure, variable temperature and multi-wavelength experiments, powder experiments automatic screening or full data collections of several in situ protein crystals, highly absorbing samples, up to 8-fold twinned samples, charge density measurements, and absolute structure determination. For 17 years, the X-Ray Crystallography Core has contributed to the training of students from across the state of Arkansas. Undergraduate research experiences stimulate students to pursue advanced degrees that lead to careers that impact human health. For other students, an undergraduate research experience help improves critical thinking and assist with writing and scientific communication skills. In 17 years, that the Core has existed, 13 future M.D, 5 future Pharm.D., one future J.D. learned how to collect diffraction data to complete their undergraduate thesis. Undergraduate research also gave students a taste of what a career in science would be like and an edge in applying for graduate schools. Seven undergraduate researchers obtained Ph.D. One such student is Leremy Colf who became the Director of Disaster Science at HHS. The structures obtained using the instrument were the heart of dissertations of 7 Ph.D. and 2 M.S. After obtaining their degrees, these students have become vital members in government, academic and private industry, e.g., FDA, Vanderbilt University Medical Center, AbbVie, Amgen and Beckman Coulter. In summary, the proposed upgrade will make the diffraction system not only the hub for X-ray diffraction within Arkansas, but also potentially for other neighboring IDeA states. To assist all users with learning how to use the software and process their data, Rigaku has committed to both train and support the department and all of the users so that they can get the structural answers to their research questions. In addition to supporting Arkansas INBRE investigators, the Core supports other investigators supported by NIH included two recent awards, R15GM140433 (PI: Chenguang Fan) and a COBRE, P20GM139768 (PI: Kyle Quinn). The upgrades will not only prevent catastrophic loss of the X-ray facility in case of the current outdated detector fails and would allow the research and teaching efforts to expand greatly on the UAF campus and across the state. With the efforts currently ongoing across Arkansas to produce novel results across multiple fields, having ready access to a diffraction center is critical. The X-Ray Crystallography Core at the University of Arkansas is on the cusp of unequivocally saying that it can be a diffraction hub for this section of the United States and will be exactly that if granted the funding.