AN ELECTRON MICROSCOPE FOR THE STUDY OF COMPLEX BIOLOGICAL ASSEMBLIES

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This subproject is one of many research subprojects utilizing the resources provided by a Shared Instrumentation Grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the grant, which is not necessarily the institution for the investigator. DESCRIPTION (provided by applicant): Cryo-electron microscopy (cryo-EM) can be used for single particle and tomographic three- dimensional reconstructions, essential tools for the study of large macromolecular assemblies and cellular organelles. In general, biological macromolecular assemblies (e.g., virus-receptor complexes and pleomorphic viruses) cannot be crystallized because of their inability to pack efficiently into a crystal lattice or because they only have short-lived temporal existence. Recent advances in cryo-electron tomography (cryo-ET) allow for the three-dimensional imaging of small cells (i.e. <1 urn in diameter) in their fully hydrated, native state. When cryo-ET is combined with cryo-sectioning of frozen-hydrated material from samples prepared using high-pressure freezing, three-dimensional information can be gathered on even larger biological specimens. These electron microscopy techniques fill the 100-5000 A scale range between X-ray crystallography and optical microscopy. The specimens for cryo-EM studies are vitrified in physiologically relevant buffer solutions and the reconstructions closely represent the native structures. Structures of the different functional states can also be determined to characterize complete dynamic processes of biological systems. At Purdue, we have had extensive experience in combining cryo-EM and crystallography. Furthermore, in recognition of some of our accomplishments, we are about to be housed in a new building exclusively for Structural Biology. We will move both of our current field-emission gun transmission electron microscopes to the new facility, including one into the planned bio-safety containment facility to study hazardous viral pathogens. The microscope located in containment will have restricted access, and when combined with the increase in use of the microscopes over the past five-year period, will necessitate the acquisition of a new instrument. For this we wish to purchase a state-of-the-art 300 kV field emission gun instrument with a stage able to maintain specimens at liquid helium temperatures. Such an instrument would allow us to push reconstructions in some favorable cases to 5 A or better. At such resolutions, secondary structural features of proteins and nucleic acids become easily recognizable. The instrument would also allow us to perform tomographic studies at about 50 A resolution on pleomorphic viruses and cell organelles that lack structural homogeneity.