RESUMO
The processes of single particle electron crystallography and three-dimensional angular reconstitution are applied to digital cryoelectron images of a macromolecular complex, the Staphylothermus marinus phosphoenolpyruvate synthase. In particular, the application of IQAD (iterative quaternionic angular determination) is exemplified in the context of more canonical approaches.
Assuntos
Microscopia Crioeletrônica/métodos , Desulfurococcaceae/ultraestrutura , Processamento de Imagem Assistida por Computador/métodos , Fosfotransferases (Aceptores Pareados)/ultraestrutura , Desulfurococcaceae/enzimologia , Modelos Moleculares , Conformação ProteicaRESUMO
The phosphoenolpyruvate synthase (EC 2.7.9.2) of the hyperthermophilic archaeon Staphylothermus marinus forms a 2.25 MDa homomultimeric complex of 24 subunits. Here, computational analysis of low-dose cryoelectron micrographs was used to ascertain that the major rotational symmetry axes were 2-fold, 3-fold, and 4-fold. These symmetry considerations were used to perform a three-dimensional reconstruction to a spatial resolution of 4 nm. This assembly has an octahedral architecture with a solvent accessible interior and with ill-defined yet seemingly flexible appendages on the periphery. This macromolecular assembly is unusually large in mass compared with most other known globular proteins--especially other identified phosphoenolpyruvate synthases which are usually dimeric--and its elaborate quaternary arrangement might represent an adaptation to its extreme environment.
Assuntos
Proteínas Arqueais/ultraestrutura , Desulfurococcaceae/enzimologia , Fosfotransferases (Aceptores Pareados)/ultraestrutura , Criopreservação , Processamento de Imagem Assistida por Computador , Substâncias Macromoleculares , Microscopia Eletrônica , Conformação ProteicaRESUMO
The phosphoenolpyruvate synthase of the hyperthermophilic archaeon Staphylothermus marinus forms an unusually large homomultimeric complex of 93 kDa subunits. Electron image analysis of negatively stained and low-dose unstained preparations showed that the complex has a single, stable characteristic view and a well-preserved core with threefold rotational symmetry. The periphery of the assembly is composed of a nebulous, possibly flexible, component. Mass measurements by scanning transmission electron microscopy yielded a molecular weight of 2250 +/- 230 kDa, confirming the well-defined nature of the structure and indicating that it is composed of 24 +/- 2.5 subunits. The stability and symmetry of the characteristic projection views suggest a polyhedral three-dimensional architecture. The novel quaternary arrangement of this enzyme might be a consequence of its adaptation to an extreme environment.
