Cryo-electron tomography and single-particle cryo-electron microscopy: Structures of Kaposi's sarcoma-associated herpesvirus and propionyl-CoA carboxylase.

摘要

Three-dimensional (3D) electron microscopy (EM) has become a powerful method in structural biology. Here we applied two 3D EM techniques, cryo-electron tomography (CryoET) and cryo-electron microscopy (CryoEM), to study the 3D structures of Kaposi's sarcoma-associated herpesvirus (KSHV) and propionyl-CoA carboxylase (PCC) respectively. The results demonstrated that 3D structures integrated with biological information can provide a better understanding of the biological process and the function of molecular machines.;KSHV belongs to the gammaherpesvirus subfamily and shares a similar multilayered architecture with other herpesviruses. Despite intensive investigation, the process of the capsid assembly of herpesvirus is still a mystery. In addition, genetic and biochemical studies have suggested the existence of a DNA-translocating portal in the herpesvirus. However, nobody had ever observed the portal in any herpesvirues. Our cryoET study revealed that the portal of KSHV is an internally localized structure, and lacks the external machinery characteristic of portals in DNA bacteriophages. We also found that the inner scaffolding of the KSHV B capsids are highly variable, which suggests these particles are intermediates at different stages of the capsid maturation pathway. This information, taken together with previous observations, has allowed us to propose a detailed capsid assembly pathway.;PCC is a biotin-dependent mitochondrial enzyme that is responsible for the chemical reaction of converting propionyl-CoA to methomenyl-CoA. Defects of PCC function can lead to a life-threatening metabolic disorder propionic acidemia (PA). The catalytic process of PCC requires a cofactor biotin to transfer the carboxyl group between the remote active sites of the PCC alpha and beta subunits. The cryoEM structure revealed that human PCC is a hollow cylinder with three spikes attached to each end. The beta subunit forms the central cylinder. Six alpha subunits, which are composed of the BC and the BCCP domains, form the spikes. Interestingly, the BC domain is arranged as monomer instead of dimer as previously believed. In addition, the linkage between the BC and BCCP domains is relatively flexible. We therefore proposed that the BCCP domain combined with biotin to form a flexible long arm, which swings between the active sites to effectively transfer the carboxyl group.