ORT: A TEST CASE WITH THE ATP-DEPENDENT CHROMATIN REMODELING COMPLEX RSC

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This subproject is one of many research subprojects utilizing the resources provided by a Center 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 Center, which is not necessarily the institution for the investigator. A variety of cellular factors are involved in regulating the dynamic behavior of chromatin, thus making specific DNA segments available, or unavailable, according to specific needs. Among them are the ATP-dependent chromatin remodeling factors, large, multisubunit complexes that use the energy from ATP hydrolysis to non-covalently modify histone-DNA contacts. Despite a large body of experimental data we do not yet understand the mechanism by which these macromolecules perform their function. We are taking a structural approach to address this problem with the goal of eventually visualizing a number of intermediates along the remodeling pathway at high resolution using cryo-electron microscopy (cryo-EM) and single-particle methods. In this particular instance, we are interested in testing the recently developed automation of data collection using the Orthogonal Tilt Reconstruction (OTR) method (which we developed) as has been incorporated into Leginon. Obtaining correct initial models for single particles by (cryo-)EM remains one of the major challenges in the field, particularly for asymmetric particles that are conformationally flexible (as it appears to be the case for these large chromatin remodelers--see (d) below). The ability to bring automated data collection to this problem would be invaluable. Furthermore, successful automated data collection of images from tilted samples under cryo conditions would allow for the application of the OTR method directly to vitrified samples, thus removing the additional complication of flattening associated with the negatively stained samples often used to obtain initial reconstructions. We have previously obtained initial reconstructions of the yeast S.cerevisiae chromatin remodeling RSC in negative stain using the OTR method and manual data collection. The goal for this project is to obtain initial reconstructions of a RSC:nucleosome complex in negative stain (and possibly vitrified) as well as an initial reconstruction of RSC under vitrified conditions in order to compare it with the one we previously obtained from a negatively stained sample.