RESUMEN
Cryo-electron tomography (cryo-ET) allows 3D volumes to be reconstructed from a set of 2D projection images of a tilted biological sample. It allows densities to be resolved in 3D that would otherwise overlap in 2D projection images. Cryo-ET can be applied to resolve structural features in complex native environments, such as within the cell. Analogous to single-particle reconstruction in cryo-electron microscopy, structures present in multiple copies within tomograms can be extracted, aligned, and averaged, thus increasing the signal-to-noise ratio and resolution. This reconstruction approach, termed subtomogram averaging, can be used to determine protein structures in situ. It can also be applied to facilitate more conventional 2D image analysis approaches. In this chapter, we provide an introduction to cryo-ET and subtomogram averaging. We describe the overall workflow, including tomographic data collection, preprocessing, tomogram reconstruction, subtomogram alignment and averaging, classification, and postprocessing. We consider theoretical issues and practical considerations for each step in the workflow, along with descriptions of recent methodological advances and remaining limitations.
Asunto(s)
Algoritmos , Microscopía por Crioelectrón/métodos , Tomografía con Microscopio Electrónico/métodos , Procesamiento de Imagen Asistido por Computador/estadística & datos numéricos , Programas Informáticos , Microscopía por Crioelectrón/instrumentación , Tomografía con Microscopio Electrónico/instrumentación , Análisis de Fourier , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/instrumentación , Imagenología Tridimensional/métodos , Modelos Moleculares , Proteínas/ultraestructura , Relación Señal-Ruido , Flujo de TrabajoRESUMEN
Transport of material within cells is mediated by trafficking vesicles that bud from one cellular compartment and fuse with another. Formation of a trafficking vesicle is driven by membrane coats that localize cargo and polymerize into cages to bend the membrane. Although extensive structural information is available for components of these coats, the heterogeneity of trafficking vesicles has prevented an understanding of how complete membrane coats assemble on the membrane. We combined cryo-electron tomography, subtomogram averaging, and cross-linking mass spectrometry to derive a complete model of the assembled coat protein complex I (COPI) coat involved in traffic between the Golgi and the endoplasmic reticulum. The highly interconnected COPI coat structure contradicted the current "adaptor-and-cage" understanding of coated vesicle formation.
Asunto(s)
Vesículas Cubiertas por Proteínas de Revestimiento/química , Proteína Coat de Complejo I/química , Factor 1 de Ribosilacion-ADP/química , Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , Proteínas Activadoras de GTPasa/química , Humanos , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
The major structural components of HIV are synthesized as a 55-kDa polyprotein, Gag. Particle formation is driven by the self-assembly of Gag into a curved hexameric lattice, the structure of which is poorly understood. We used cryoelectron tomography and contrast-transfer-function corrected subtomogram averaging to study the structure of the assembled immature Gag lattice to approximately 17-A resolution. Gag is arranged in the immature virus as a single, continuous, but incomplete hexameric lattice whose curvature is mediated without a requirement for pentameric defects. The resolution of the structure allows positioning of individual protein domains. High-resolution crystal structures were fitted into the reconstruction to locate protein-protein interfaces involved in Gag assembly, and to identify the structural transformations associated with virus maturation. The results of this study suggest a concept for the formation of nonsymmetrical enveloped viruses of variable sizes.
