This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.ABSTRACT: An important application of cryo-electron tomography is to identify and determine the orientation and conformation of large macromolecular assemblies in their native cellular environment. This requires the development of algorithms that can efficiently detect target structural motifs within noisy tomographic 3-D density maps, using probe structures provided by single-particle cryo-EM or x-ray crystallography. Dr. Renken is continuing to work on improved methods for motif search using RAMOS (Rapid Motif Search: Rath et al., 2004, J. Struct. Biol. 145, 84-90). RAMOS is an algorithm that uses local variance in order to calculate the locally normalized cross-correlation function between a small motif and a larger volume. The method is being tested using tomographic reconstructions of frozen-hydrated triad junctions from skeletal muscle. The challenge is to determine the location and orientation of ryanodine receptors (RyR) on vesicles of sarcoplasmic reticulum (SR). The motif used for searching is a 3-nm-resolution map of the RyR derived from single-particle reconstruction. Last year, the position and orientation of 12 receptors in a disordered membrane array were determined and an average calculated. While considerably lower in resolution (8 nm) than those routinely obtained from thousands of detergent-extracted particles, this in situ RyR average has an important advantage, namely the presence of the SR membrane itself and neighboring structures such as calsequestrin in the SR lumen. In effect, these in situ averages will describe not only the RyR but the 'calcium release unit' of the triad junction.
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