RESUMEN
High-resolution structural information is essential to understand protein function. Protein-structure determination needs a considerable amount of protein, which can be challenging to produce, often involving harsh and lengthy procedures. In contrast, the several thousand to a few million protein particles required for structure determination by cryogenic electron microscopy (cryo-EM) can be provided by miniaturized systems. Here, we present a microfluidic method for the rapid isolation of a target protein and its direct preparation for cryo-EM. Less than 1 µL of cell lysate is required as starting material to solve the atomic structure of the untagged, endogenous human 20S proteasome. Our work paves the way for high-throughput structure determination of proteins from minimal amounts of cell lysate and opens more opportunities for the isolation of sensitive, endogenous protein complexes.
Asunto(s)
Microscopía por Crioelectrón/métodos , Procesamiento de Imagen Asistido por Computador/estadística & datos numéricos , Complejo de la Endopetidasa Proteasomal/ultraestructura , Subunidades de Proteína/química , Biotinilación , Microscopía por Crioelectrón/instrumentación , Células HeLa , Humanos , Imagenología Tridimensional , Fragmentos Fab de Inmunoglobulinas/química , Técnicas Analíticas Microfluídicas/métodos , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/aislamiento & purificación , Conformación Proteica , Subunidades de Proteína/aislamiento & purificación , VitrificaciónRESUMEN
One of the largest membrane protein families in eukaryotes are G protein-coupled receptors (GPCRs). GPCRs modulate cell physiology by activating diverse intracellular transducers, prominently heterotrimeric G proteins. The recent surge in structural data has expanded our understanding of GPCR-mediated signal transduction. However, many aspects, including the existence of transient interactions, remain elusive. We present the cryo-EM structure of the light-sensitive GPCR rhodopsin in complex with heterotrimeric Gi. Our density map reveals the receptor C-terminal tail bound to the Gß subunit of the G protein, providing a structural foundation for the role of the C-terminal tail in GPCR signaling, and of Gß as scaffold for recruiting Gα subunits and G protein-receptor kinases. By comparing available complexes, we found a small set of common anchoring points that are G protein-subtype specific. Taken together, our structure and analysis provide new structural basis for the molecular events of the GPCR signaling pathway.