Electron Microscopic Studies of Macromolecular Complexes

The single particle method of electron microscopic reconstruction is used to study several protein structures including papillomavirus E1 helicase, bacteriophage T7 gene 4 helicase-primase complex, E. coli RuvB branch migration protein, dynamin lipid-tube and archaeal MCM helicase. Papillomavirus E1 helicase has multiple functions and is involved in the early stage of virus genome replication in the host cell. Its reconstruction shows a very different domain arrangement compared to SV40 large T antigen, even though they are predicted to be homologs by sequence analysis, secondary structure prediction and atomic structure comparison of the DNA binding domain and the helicase domain. Bacteriophage T7 gene 4 protein contains a helicase and primase domain. Reconstructions display the structural dynamics of the primase domain, which is consistent with its crystal structure.
The atomic details of the RuvB hexamer are revealed by docking the crystal structure of the RuvB monomer into the hexameric reconstruction. The result agrees with several mutational and biochemical studies, and offers a better model than the one based on the hexameric crystal structures of a RuvB homolog. Electron microscopic reconstruction is a powerful tool in studying very large macromolecular complexes. It provides the first three-dimensional model for the mechanism of dynamin lipid-tube constriction, which is essential for receptor-mediated endocytosis. The diameter of a dynamin lipid-tube changes from 50 nm to 40 nm during GTP hydrolysis and the surface area of the underlying lipid-tube is reduced by half due to the bending of the stalk region of dynamin, which may be the driving force for the fission of the neck of the endocytotic vesicles. Finally, MCM helicase is responsible for replication initiation and prevents multiple replication of the genome in the cell cycle. An analysis of archaeal MCM filaments and its N-terminal fragment shows a large conformational change of the Nterminal domains of archael MCM. It provides strong support for the domain-push model explaining the phosphorylation bypass phenotype of yeast MCM bob1 mutation.