1. Draft crystal structure of the vault shell at 9-A resolution
- Author
-
Stuart A. Sievers, David Eisenberg, Valerie A. Kickhoefer, Leonard H. Rome, Daniel H. Anderson, and Petsko, Gregory A
- Subjects
Models, Molecular ,Electron density ,QH301-705.5 ,Molecular Sequence Data ,Shell (structure) ,Bioengineering ,Geometry ,02 engineering and technology ,Medical and Health Sciences ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Models ,Major vault protein ,Animals ,Biology (General) ,Molecular Biology ,Vault (organelle) ,030304 developmental biology ,Ribonucleoprotein ,Vault Ribonucleoprotein Particles ,0303 health sciences ,General Immunology and Microbiology ,biology ,Base Sequence ,Agricultural and Veterinary Sciences ,General Neuroscience ,Resolution (electron density) ,Cryoelectron Microscopy ,Molecular ,Biological Sciences ,021001 nanoscience & nanotechnology ,Protein tertiary structure ,Recombinant Proteins ,Rats ,Liver ,biology.protein ,Substructure ,0210 nano-technology ,General Agricultural and Biological Sciences ,Crystallization ,Research Article ,Developmental Biology - Abstract
Vaults are the largest known cytoplasmic ribonucleoprotein structures and may function in innate immunity. The vault shell self-assembles from 96 copies of major vault protein and encapsulates two other proteins and a small RNA. We crystallized rat liver vaults and several recombinant vaults, all among the largest non-icosahedral particles to have been crystallized. The best crystals thus far were formed from empty vaults built from a cysteine-tag construct of major vault protein (termed cpMVP vaults), diffracting to about 9-Å resolution. The asymmetric unit contains a half vault of molecular mass 4.65 MDa. X-ray phasing was initiated by molecular replacement, using density from cryo-electron microscopy (cryo-EM). Phases were improved by density modification, including concentric 24- and 48-fold rotational symmetry averaging. From this, the continuous cryo-EM electron density separated into domain-like blocks. A draft atomic model of cpMVP was fit to this improved density from 15 domain models. Three domains were adapted from a nuclear magnetic resonance substructure. Nine domain models originated in ab initio tertiary structure prediction. Three C-terminal domains were built by fitting poly-alanine to the electron density. Locations of loops in this model provide sites to test vault functions and to exploit vaults as nanocapsules., Author Summary Vaults are large barrel-shaped particles found in the cytoplasm in all mammalian cells, which may function in innate immunity. As naturally occurring nanoscale capsules, vaults may be useful objects to engineer as delivery vehicles. In this study, we propose an atomic structure for the thin outer shell of the vault. Using x-ray diffraction and computer modeling, we have inferred a draft atomic model for the major vault protein, which forms the shell-like enclosure of the vault. The shell is made up of 96 identical protein chains, each of 873 amino acid residues, folded into 14 domains. Each chain forms an elongated stave of half the vault, as well as the cap of the barrel-like shell. Our draft atomic model is essentially an atomic-level model for the entire 9.3-MDa vault shell, which offers a guide for protein engineering to test vault functions and to exploit vault particles as nanocapsules., A draft atomic structure has been determined for the 9.3-MDa protein shell of the vault cytoplasmic particle, revealing stave-like polypeptides forming the barrel-like structure of the vault.
- Published
- 2007