Cryo-EM reconstruction of recombinant and tissue derived vaults and other macromolecular assemblies

Vaults are large ribonucleoprotein particles that are conserved in higher eukaryotes and have been associated with the multidrug resistance phenotype in human cancer cells. They consist of three proteins, the major vault protein (MVP), vault poly-ADP ribose polymerase (VPARP), and telomerase associated protein (TEP1), as well as untranslated vRNA. Vaults can be produced recombinantly in insect cells with MVP alone, or in combination with VPARP and/or TEP1. Knockout mice lacking TEP 1 have also been produced for structural and functional studies. Cryo-EM imaging has proven to be a powerful way to analyze the vault structure at moderate resolution. Fold recognition methods have been applied to interpret the cryo-EM density.; This thesis presents cryo-EM reconstructions of numerous recombinant vaults with varying protein compositions and with different peptide tags added to the N-terminus of MVP; wild-type mouse vaults for comparison with vaults isolated from TEP1 knockout mice; and wild-type sea urchin vaults. The recombinant vault reconstructions demonstrate that MVP alone can form particles with the same morphology as naturally produced vaults. A comparison of recombinant vaults with various length peptide tags provides evidence that the N-termini of MVP meet at the central constriction of the particle. A difference map analysis indicates that TEP1 is localized within the cap region of the vault. Fold models for several domains of TEP1, including TROVE, VWA, and NACHT, were identified. In addition, a total of 21 WD40 repeats were found at the C-terminus of TEP1, five more than were identified earlier by sequence analysis. The reconstruction of the sea urchin vault shows that they have a similar exterior structure to rat and mouse vaults even though they have a more complex protein composition.; Another macromolecular assembly that was imaged by cryo-EM was embryonic alpha-crystallin. Comparison of the embryonic form with mature alpha-crystallin indicates that the multimeric protein structure becomes more variable with age probably because of numerous post-translational modifications.; These studies demonstrate the potential of cryo-EM imaging for studying the structure of large macromolecular assemblies that are not suitable for other structural determination methods.