The Biological Science Imaging Resource (BSIR) at Florida State University (FSU) is the focal point for cryoelectron microscopy and 3-D electron microscopy in the state of Florida and the Southeast. No other University facility has a comparable emphasis or facilities. This application requests funds to replace and upgrade the center piece of this facility, an FEI CM 300-FEG electron microscope with a Titan-Krios, a robotic electron microscope with enhanced capabilities for cryoEM and electron tomography. Our CM 300-FEG microscope is 15 years old and is showing its age both in terms of down time and our ability to implement instrumentation and software developments produced by others in the field. The instrument lacks an imaging filter which is crucial for work that now involves thick specimens, and will become increasing problematic for retrofit of new developments in electron microscopy such as phase plates and aberration correctors. The requested Titan-Krios will support the research of the major BSIR users, Drs. Kenneth Taylor, Kenneth Roux and Thomas Roberts, new faculty member Dr. Scott Stagg and visitors from the lab of a long standing collaborator, Dr. Michael Chapman, who recently moved to Oregon Health & Science University. Three minor projects are described that will benefit from access to a modern electron microscope. The Titan-Krios will also be able to support remote users, making it possible to offer its extensive capabilities conveniently to users outside of the university and become a true research resource for the Southeast. The study of thin, vitreous, frozen hydrated suspensions, both using electron tomography and single particle methods is a large and growing cryoEM research emphasis at FSU, but obtaining sufficient data, either as tilt series or projections is currently a bottleneck in a number of projects. The robotic capabilities of the Titan-Krios as well as the ability to run state of the art automated EM software, such as Leginon, will alleviate this bottleneck. Electron tomography is a major emphasis of three applicant groups while the other two are beginning to explore the possibilities. The dual axis tomography capabilities of the Titan-Krios will improve both throughput and quality of reconstructions obtained from both frozen hydrated as well as rapidly frozen, freeze substituted, plastic embedded biological specimens. The Titan-Krios will support NIH funded research in (1) the fundamental mechanism of force generation in a myogenic muscle, which has health relevence to cardiac muscle function; (2) smooth muscle myosin regulation, which has health relevance in hypertension; (3) structures involved in cell migration, which has health relevence in cancer; (4) the structure of HIV and SIV viruses which are fundamental to understanding AIDS; (5) the development of Adeno Associated Virus as a gene therapy vector and (6) NIH related research into the structure of COPII vesicles, which are implicated in defects of cargo trafficking including chylomicron retention disease and cranio-lenticulo-sutural displasia. ? ? ?
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