Summary Macromolecular structure determination at atomic resolution via X-ray crystallography is arguably one of the greatest technical achievements in the life sciences over the last century. By shedding light on molecular mechanisms essential for life, the technique has dramatically deepened our understanding of health and disease and accelerated the development of drugs that are more selective, potent, and safe. The story of protein crystallography at Purdue began in 1964 with the recruitment of Michael Rossmann, a pioneer in the field. A formal biophysics training program was instituted in 1989 with an NRSA Institutional Predoctoral Training Grant, coinciding with the award of a multi-million dollar Markey Foundation grant, which provided funds for hiring additional faculty and the X-ray generators currently in the existing X-ray facility. Armed with excellent facilities and expert faculty dedicated to advancing molecular biophysics and the training of junior scientists, Purdue produced many successful investigators including Hao Wu (Harvard), Liang Tong (Columbia), Marvin Hackert (UT Austin), and Jack Johnson (Scripps). John Tesmer, the PI of this proposal, is likewise a Purdue trainee. Purdue has also served as an important hub for innovation in the field. For example, Dr. Rossmann and Dr. Janet Smith were key contributors to the techniques of molecular replacement and MAD phasing, respectively, which are now the two most commonly used phasing techniques world-wide. A new era in Purdue structural biology has begun. In 2009, the university opened Hockmeyer Hall of Structural Biology, a 30 million dollar facility and the new home of the Macromolecular Crystallography core. Purdue reaffirmed its commitment to structural biology by updating the core with the 2019 purchase of a RockImager Duo crystal hotel and imaging system, and an Oxford Cryosystems 800 Series liquid nitrogen cooler. A reboot of our T32 Molecular Biophysics Training Program (funded 2019-24) now supports 7 trainees per year (Tesmer PI). The specific aim of this proposal is to fulfill a critical need by replacing our aging ~30 year old X-ray generators, which can no longer be repaired, with a modern, reliable, and cost effective MicroMax-007 HF rotating anode generator coupled to a synchrotron quality, Dectris Eiger2 hybrid photon counting detector. The generator and its advanced optics will provide a brilliant, small 90 µm X-ray beam at the crystal, optimizing signal-to-noise for small samples. The large 155x163 mm detector will be ideal for weak reflections, with small 75 µm pixels to resolve closely spaced reflections. A 4- circle kappa goniometer will add flexibility in data collection. The new setup will improve X-ray flux >100-fold, and the detector sensitivity is about 10 times greater than imaging plates, thereby improving our overall performance by about three orders of magnitude and enabling the collection of superior diffraction data from even the most challenging cry...