RESUMO
Structure determination of helical specimens commonly requires datasets from thousands of micrographs often obtained by automated cryo-EM data acquisition. Interactive tracing of helical assemblies from such a number of micrographs is labor-intense and time-consuming. Here, we introduce an automated tracing tool MicHelixTrace that precisely locates helix traces from micrographs of rigid as well as very flexible helical assemblies with small numbers of false positives. The computer program is fast and has low computational requirements. In addition to helix coordinates required for a subsequent helical reconstruction work-flow, we determine the persistence length of the polymer ensemble. This information provides a useful measure to characterize mechanical properties of helical assemblies and to evaluate the potential for high-resolution structure determination.
Assuntos
Algoritmos , Automação/métodos , Microscopia Crioeletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Reprodutibilidade dos TestesRESUMO
p62/SQSTM1 is an autophagy receptor and signaling adaptor with an N-terminal PB1 domain that forms the scaffold of phase-separated p62 bodies in the cell. The molecular determinants that govern PB1 domain filament formation in vitro remain to be determined and the role of p62 filaments inside the cell is currently unclear. We here determine four high-resolution cryo-EM structures of different human and Arabidopsis PB1 domain assemblies and observed a filamentous ultrastructure of p62/SQSTM1 bodies using correlative cellular EM. We show that oligomerization or polymerization, driven by a double arginine finger in the PB1 domain, is a general requirement for lysosomal targeting of p62. Furthermore, the filamentous assembly state of p62 is required for autophagosomal processing of the p62-specific cargo KEAP1. Our results show that using such mechanisms, p62 filaments can be critical for cargo uptake in autophagy and are an integral part of phase-separated p62 bodies.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Transporte/química , Proteína Sequestossoma-1/química , Proteína Sequestossoma-1/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arginina/química , Autofagia/fisiologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Células HeLa , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Lisossomos/metabolismo , Polimerização , Conformação Proteica , Domínios Proteicos , Proteína Sequestossoma-1/genéticaRESUMO
This chapter describes the recombinant overexpression of the canonical selective autophagy receptor p62/SQSTM1 in E. coli and affinity purification. Also described is the method to induce p62 filament assembly and their visualization by negative stain electron microscopy (EM). In cells, p62 forms large structures termed p62 bodies and has been shown to be aggregation prone. This tendency to aggregate poses problems for expression and purification in vitro, which is a prerequisite for structural analysis. Here, we describe the method to express and purify soluble p62, using the solubility tag, MBP, in conjunction with autoinduction. Furthermore, we describe the protocol to assemble p62 into filaments by controlling the ionic strength of its buffer, as well as the preparation of negative stain EM grids to visualize the filaments. In vitro formed p62 filaments can be used to study receptor cargo interactions in minimal reconstituted autophagy model systems.