DESCRIPTION (provided by applicant): The specific Cryo-Electron Microscope (cryoEM) to be acquired is a JEOL JEM-2100 that is a 200 kV Transmission Electron Microscope (TEM), with a Lanthanum Boride (LaB6) emitter, a full Cryo package and a stage-tilting capability for tomography. This TEM is one of the easiest to operate and maintain making it the choice of many cryo TEM facilities as a first instrument to acquire. It is often retained as a training instrument when facilities expand because it has a reputation for versatility in a multi-user environment and is relatively inexpensive to repair and maintain. It functions better than comparable instruments to fulfill the basic needs of a general research environment. A structural study offers a powerful tool of direct visualization that can guide and complement other research approaches. CryoEM allows three-dimensional (3D) imaging at the subcellular level, filling a gap between the atomic resolution provided by NMR and Xray, and visualization by light microscopy of larger entities such as bacteria, whole cells and organelles. To obtain 3-D structures from cryoEM data, single particle reconstructions rely on collecting different two dimensional views of the same object that are then reconstructed into a 3-D map. Tomography focuses on a single object and obtains the different views necessary for reconstruction by tilting the stage and taking a series of 2-D images. The subjects for visualization by the PI and CoPI's of this acquisition grant range broadly. Included are multi-protein complexes and two different DNA-protein complexes. The small polypeptide binding sites will be visualized on an RNA polymerase. The assembly intermediates of Rous sarcoma virus, the membranous webs formed during [picornavirus] replication, and a viral RNA replication initiation complex will be examined. Virus-receptor complexes will be studied, including the structure of a virus interacting with a receptor and a co-receptor simultaneously. [Using tomography the number of melanosomes will be quantified and the 3D structure refined in mutant and wild type zebrafish. Finally, the podocyte microenvironment and structure in diabetic nephropathy will be visualized by tomography.] The diversity of these projects, each chosen because of the likelihood of success showcases the powerful application of cryo-electron microscopy techniques. We identify 9 labs and describe 12 projects ready for data collection and data processing using cryoEM single particle reconstruction and tomography techniques.
|Effective start/end date||4/1/12 → 3/31/13|
- National Institutes of Health: $600,000.00
Rous sarcoma virus
DNA-Directed RNA Polymerases