We are requesting funds for a 200 kV electron microscope (EM) with a field-emission gun (FEG) and scanning unit (STEM), a liquid-nitrogen-cooled specimen holder and a state-of-the-art large-format electronic image detector (CMOS camera). The primary reason for the need of this new instrument is the increased demand on our current instruments from new projects and users, including a recently hired junior faculty member. The increased need also comes from existing projects that require collection of large data sets. The new instrument will be housed in our existing Brandeis EM facility and expand its capacity and capabilities. Specifically, the STEM unit will enable us to perform mass-per-length measurements on amyloid fibrils, a key characteristic of these fibrils. The large-format detector offers an alternative to recording data on film, thus accelerating research. Projects benefiting from the new instrument are broad in scope and include the 3D structural analysis of amyloid fibrils formed by Alzheimer's A2 peptide, the structure of cytoplasmic dynein and conformational changes associated with its power stroke, structural studies of the actin nucleator formin bound to regulators and growing actin filaments, viral cell entry, and the molecular organization of kinetochores. All of these projects address serious disorders associated with human disease, or the mechanism of prominent human pathogens (viruses). The role of A2 fibrils in Alzheimer's disease is not well understood and fibrils, as well as smaller aggregate may lead to the neuronal death observed in the course of the disease. The molecular structure of a number of morphologies of these fibrils will be determined by cryo-EM. This will shed light on their role, as well as the structure of the smaller aggregates, which act as fibril precursors. Cytoplasmic dynein plays a major role in cell division, signaling, cell shape, and polarized cell growth. The structure of dyneins in different states and bound to microtubules will be determined using electron cryo-tomography (cryo-ET). Many critical cellular functions also depend on the precise control of actin assembly and disassembly, for example by formins. The structure of formins in complex with several formin regulators will be determined by single particle cryo-EM. Furthermore, the precise mechanism of actin nucleation by formins will be investigated by cryo-ET of formins attached to growing actin filaments. Viral cell entry is one of the key events in the viral life cycle, and different viruses employ different strategies. Cell entry will be studied using a model system based on """"""""small virus-like particles"""""""" isolated by expression of the flavivirus prM and E proteins. These will be visualized by single particle cryo-EM, combined with a single-particle, fluorescence-based assay. Finally, the formation of kinetochores is central to mitotic cell division and the faithful separation of the sister chromatids from each other. A better understanding of the 3D structure of kinetochores will be achieved by combining X-ray crystallographic studies with cryo-EM of smaller subassemblies (a parallel effort to visualize intact kinetochores using electron tomography is underway at Harvard Medical School).
