Structural basis of p62/SQSTM1 helical filaments and their role in cellular cargo uptake.

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.

Recombinant Expression, Purification, and Assembly of p62 Filaments.

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).

Automated tracing of helical assemblies from electron cryo-micrographs.

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.