RNA location and modeling of a WD40 repeat domain within the vault
The vault complex is a ubiquitous 13-MDa ribonucleoprotein assembly, composed of three proteins (TEP1, 240 kDa; VPARP, 193 kDa; and MVP, 100 kDa) that are highly conserved in eukaryotes and an untranslated RNA (vRNA). The vault has been shown to affect multidrug resistance in cancer cells, and one particular component, MVP, is thought to play a role in the transport of drug from the nucleus. To locate the position of the vRNA, vaults were treated with RNases, and cryo-electron microscopy (cryo-EM) was performed on the resulting complexes. Using single-particle reconstruction techniques, 3,476 particle images were combined to generate a 22-[Angstrom capital A, ring]-resolution structure. Difference mapping between the RNase-treated vault and the previously calculated intact vault reconstructions reveals the vRNA to be at the ends of the vault caps. In this position, the vRNA may interact with both the interior and exterior environments of the vault. The finding of a 16-fold density ring at the top of the cap has allowed modeling of the WD40 repeat domain of the vault TEP1 protein within the cryo-EM vault density. Both stoichiometric considerations and the finding of higher resolution for the computationally selected and refined “barrel only” images indicate a possible symmetry mismatch between the barrel and the caps. The molecular architecture of the complex is emerging, with 96 copies of MVP composing the eightfold symmetric barrel, and the vRNA together with one copy of TEP1 and four predicted copies of VPARP comprising each cap.(Received January 25 2000)
(Revised February 17 2000)
(Accepted March 20 2000)
Key Words: [beta]-propeller; cryo-electron microscopy; difference mapping; image reconstruction.
c1 Reprint requests to: Phoebe L. Stewart, Department of Molecular and Medical Pharmacology, Crump Institute for Biological Imaging, UCLA School of Medicine, A-324 CIBI, Box 951770, 10833 LeConte Avenue, Los Angeles, California 90095-1770, USA; e-mail: email@example.com.