This proposal concerns the acquisition of a state-of-the-art electron microscope by the newly created Skirball Institute at New York University Medical Center for determination of protein structure at nearatomic resolution. The microscope has a Schottky field emission gun and is capable of operating at 200kV. In addition, accessories will make it possible to image frozen-hydrated biological specimens at high resolution. Finally, digital and analogue interfaces will allow control of the microscope by external computers and will be used to customize the operating modes. Two projects are described that use methods of electron crystallography to study the molecular structure of Ca2+-ATPase from sarcoplasmic reticulum and of the anion transporter from erythrocytes (Band 3). In the case of Ca2+-ATPase, thin, multilamellar crystals diffract to 3.5 resolution and 3-D data will be collected by tilting specimens and recording both electron diffraction and images. The same strategy will be used for 2-D crystals of Band 3, which in initial studies have produced optical diffraction to 12 . In both cases, the aim is for 3-D reconstruction at ~7 resolution, from which elements of secondary structure will be identified and related to the extensive set of biochemical and biophysical data for each protein. For Ca2+-ATPase, the relatively strong electron diffraction provides hope for a reconstruction at higher resolution, which may eventually allow tracing of the peptide chain. Both of these projects also involve tubular crystals of their respective proteins, which provide structural data at more moderate resolutions (~15 ), but which offer the opportunity to study conformational changes of the molecules. Such conformational changes are integral to the respective mechanisms of ion transport and elucidation of the corresponding structures will be essential for fully understanding these mechanisms. A collaborative project with the Neurobiology Program at the Skirball Institute is also described. This involves immunoelectron microscopy of tissue sections in an attempt to understand the sequence of signals that lead to formation of a neuromuscular junction during development. In addition, other investigators are being recruited into the Neurobiology Program with interests in structure/function relationships of voltagegated ion channels. Ultimately, active collaborations between the electron microscopists and the neurobiologists are likely to engender a crystallographic project involving these channels. The building of a structural biology group at the core of a major new research institute at New York University Medical Center will not only provide extensive training opportunities for the graduate students, but also bring New York University to the forefront of this important field. The lack of structural information for membrane proteins represents an acknowledged problem for the basic research community and all of the projects to be pursued on the requested electron microscope would specifically address this problem. In addition, the unique capabilities of this microscope for high resolution imaging of biological molecules would make it a major resource for structural biologists throughout the northeastern United States.
