The overall goal of this project is to establish the experimental restraints necessary to construct a molecular model for SPB core that extends from atomic structures of the individual components to their interactions with each other and their overall position in the MTOC where the information will be assembled into a cohesive structure by the computational core. This structural study complements the investigation of the structure of the y-tubulin complexes that constitute the inner and outer plaques that will be performed by Project 2 (Agard). The experimental plan includes three complementary approaches.
The first aim (Rayment) begins with high resolution X-ray structural determinations of the individual components of the SPB and proceeds in a logical progression from the structures of the proteins and domains that constitute the central plaque which is embedded in the nuclear envelope (Spc110-C, calmodulin, Spc29, and Spc42-N) through a structural determination of the Intermediate layers 2 and 1 (Spc42-C, Cnm67, Nud1, and Spc72-C). Intermediate Layer 1 forms the bridge to the y-tubulin (Tub4) complex in the outer plaque so that this project interfaces and complements the study of the y-tubulin described in Project 2. At this time all of the proteins in the SPB core have been expressed in a soluble form suitable for structural or biophysical study and more than half of them have been crystallized.
The second aim i s directed towards establishing a molecular envelope for the native SPB into which the high-resolution structures can be docked or modeled (Agard). This will be established through cryo electron tomography (cryoET) and subvolume averaging of entire isolated SPB. This will provide an unbiased 3D framework of the entire SPB at an intermediate resolution of about 20 ?. It will reveal the domain organization within each layer and the interaction of major SPB components between layers. Protein-tagging will be used to locate and orient individual proteins within the maps.
The third aim (Davis) is directed towards generating a new set of distance restraints between individual components that are needed to combine the information from the previous specific aims. Two approaches will be used. First, the prior FRET analysis that established the current arrangement of SPB components in the core will be extended for proteins in the central plaque. Second, a new set of high-resolution distances will be established through crosslinking analysis of native SPB assemblies. These structural investigations will provide detailed structures of the individual components and the manner in which they are arranged in the SPB. Thus, it will establish air of the information necessary for the computational core to generate a pseudo atomic model for the SPB. In turn, hypotheses that arise from this three-dimensional model will be tested through biochemical, genetic and cell biological studies in Projects 3 and 4 (Davis, Winey and Rayment). It will also allow the mechanical strengths of the microtubule-SPB attachments measured in Project 5 (Asbury) to be interpreted in molecular terms.
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