RESUMEN
We have used Leginon, a fully automatic system capable of acquiring cryo-electron micrographs, to collect data of single particles, specifically of the AAA ATPase p97. The images were acquired under low-dose conditions and required no operator intervention other than the initial setup and periodic refilling of the cold-stage dewar. Each image was acquired at two different defocus values. Two-dimensional projection maps of p97 were calculated from these data and compared to results previously obtained using the conventional manual data collection methods to film. The results demonstrate that Leginon performs as well as an experienced microscopist for the acquisition of single-particle data. The general advantages of automation are discussed.
Asunto(s)
Adenosina Trifosfatasas/química , Microscopía por Crioelectrón/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Proteínas Nucleares/química , Animales , Bovinos , Microscopía por Crioelectrón/instrumentación , Microscopía por Crioelectrón/tendencias , Procesamiento Automatizado de Datos , Procesamiento de Imagen Asistido por Computador/instrumentación , Procesamiento de Imagen Asistido por Computador/normas , Tamaño de la Partícula , Conformación ProteicaRESUMEN
AAA ATPases play central roles in cellular activities. The ATPase p97, a prototype of this superfamily, participates in organelle membrane fusion. Cryoelectron microscopy and single-particle analysis revealed that a major conformational change of p97 during the ATPase cycle occurred upon nucleotide binding and not during hydrolysis as previously hypothesized. Furthermore, our study indicates that six p47 adaptor molecules bind to the periphery of the ring-shaped p97 hexamer. Taken together, these results provide a revised model of how this and possibly other AAA ATPases can translate nucleotide binding into conformational changes of associated binding partners.
Asunto(s)
Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas de Transporte Vesicular , Adenosina Difosfato/metabolismo , Adenosina Trifosfatasas/ultraestructura , Adenilil Imidodifosfato/metabolismo , Animales , Proteínas Portadoras/química , Proteínas Portadoras/ultraestructura , Bovinos , Microscopía por Crioelectrón , Procesamiento de Imagen Asistido por Computador , Proteínas de la Membrana/metabolismo , Ratones , Modelos Moleculares , Proteínas Sensibles a N-Etilmaleimida , Proteínas Nucleares/ultraestructura , Unión Proteica , Conformación Proteica , Proteínas SNARERESUMEN
African swine fever (ASF) virus is a large DNA virus that shares the striking icosahedral symmetry of iridoviruses and the genomic organization of poxviruses. Both groups of viruses have a complex envelope structure. In this study, the mechanism of formation of the inner envelope of ASF virus was investigated. Examination of thin cryosections by electron microscopy showed two internal membranes in mature intracellular virions and all structural intermediates. These membranes were in continuity with intracellular membrane compartments, suggesting that the virus gained two membranes from intracellular membrane cisternae. Immunogold electron microscopy showed the viral structural protein p17 and resident membrane proteins of the endoplasmic reticulum (ER) within virus assembly sites, virus assembly intermediates, and mature virions. Resident ER proteins were also detected by Western blotting of isolated virions. The data suggested the ASF virus was wrapped by the ER. Analysis of the published sequence of ASF virus (R. J. Yanez et al., Virology 208:249-278, 1995) revealed a reading frame, XP124L, that encoded a protein predicted to translocate into the lumen of the ER. Pulse-chase immunoprecipitation and glycosylation analysis of pXP124L, the product of the XP124L gene, showed that pXP124L was retained in the ER lumen after synthesis. When analyzed by immunogold electron microscopy, pXP124L localized to virus assembly intermediates and fully assembled virions. Western blot analysis detected pXP124L in virions isolated from Percoll gradients. The packaging of pXP124L from the lumen of the ER into the virion is consistent with ASF virus being wrapped by ER cisternae: a mechanism which explains the presence of two membranes in the viral envelope.