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 electron camera on the center piece of this facility, an FEI Titan-Krios electron microscope with a direct electron camera, a revolutionary camera that is far superior to any previous image recording method. We will replace an existing Gatan Ultrascan camera with a Gatan K2 Summit camera. The requested K2 Summit will support the research of the major BSIR users, Drs. Kenneth Taylor, Scott Stagg, Hong Li, and new faculty member Dr. Elizabeth Stroupe. It will also provide visitors to the BSIR from outside FSU access to a state of the art facility. Two new investigators outside of FSU have provided projects requiring access to a modern electron microscope with a direct electron camera. The study of thin, vitreous, frozen hydrated suspensions, using both electron tomography and single particle methods is a large and growing cryoEM research emphasis worldwide. The robotic capabilities of the Titan-Krios with its ability to run state of the art automated EM software, such as Leginon, are well adapted to this need, but ultimately, the quality of the recording medium limits the quality of the final result. The poor MTF of today's CCD cameras is offset somewhat by recording images at higher magnification, but this reduces the field of view requiring even more images. Beam induced specimen motion is recognized as a fundamental problem that can be solved by rapid exposure times, known as movie mode, and local (or global) corrections for particle motion. Movie mode images will be crucial for electron tomography, which relies on images taken at high tilt angles for best results. Such images are prone to beam induced specimen motion perpendicular to the specimen plane, a problem that would be reduced by correcting for motion in 25 msec image recording times. 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) myosin motor activity, which has health relevance in multiple diseases;(3) transformation of bacteria by bacteriophage which leads to multidrug resistance;(4) the structure of COPII vesicles, which are implicated in defects of cargo trafficking including chylomicron retention disease and cranio-lenticulo-sutural displasia;(5) The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) which confer adaptive immunity of bacteria against invasive genetic elements and (6) NIH related research into the structure and function of sulfite reductases.
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