PROJECT SUMMARY/ABSTRACT The University of Michigan (U-M) has established a cutting-edge cryo-electron microscopy (cryo-EM) facility with multiple high-resolution microscopes equipped with the newest direct electron detectors (DEDs). The facility uses automated data acquisition and streamlined image processing protocols, some developed at U-M, which allows for high-throughput structural determination of biological molecules using both single-particle and cryo-electron tomography (cryo-ET) strategies. The combination of improved cameras and automated data collection now makes it routine for users to rapidly collect the data required to determine sub-3.5 Å structures, resolutions that allow for direct model building in the resulting density maps. However, each cryo-EM dataset requiring terabytes of storage and thousands of CPU (Central Processing Unit) or GPU (Graphics Processing Unit) hours on computer systems equipped with large amounts of random-access memory (RAM). This makes high-throughput data collection a new bottleneck in the cryo-EM pipeline. The ability to effectively store, process, and analyze the increasingly massive datasets is essential for this process. Thus, the ability to quickly collect large datasets, combined with a rapidly expanding campus-wide user base, means that access to computational resources now represents a major limiting factor for the success of many U-M cryo-EM projects. The computational resources for cryo-EM at the University of Michigan, housed at the Life Sciences Institute (LSI), were originally funded in part through an S10 grant to the university and will reach their end of life by May 2021 when they will no longer be supported by the vendor. This proposal requests funds to replace the aging computation and storage system, as well as integrate a powerful compute cluster that has nodes with enough memory to efficiently determine atomic resolution structures of large particles (> 1,000 pixel box sizes). This upgraded computation cluster will include 1,248 Intel CPU cores, 16 NVIDIA Quadro RTX 8000 GPUs (Graphic Processing Units), two large memory nodes, and 820 TB of storage, which will be combined and integrated with some of our newer CPU/GPU compute nodes that will remain online. Installation of this resource is essential for: 1) the U-M cryo-EM facility ability to ramp up data collection on four high-resolution cryo-electron microscopes with “on-the-fly” processing; 2) image processing of large heterogeneous cryo-EM datasets (+1,000,000 particles); and 3) compute nodes with enough memory to accommodate the structural analysis of large macromolecular complexes, such as viruses and bacterial secretion systems, and the large volumes used for in situ cryo-ET analysis. The computational infrastructure proposed here will significantly enhance state-of-the-art cryo-EM structure determination at U-M, facilitating the scientific progress of numerous NIH supported investigators, as well as a cohort of talented junio...