Visualization of the Cdc48 AAA+ ATPase protein unfolding pathway.

The Cdc48 AAA+ ATPase is an abundant and essential enzyme that unfolds substrates in multiple protein quality control pathways. The enzyme includes two conserved AAA+ ATPase motor domains, D1 and D2, that assemble as hexameric rings with D1 stacked above D2. Here, we report an ensemble of native structures of Cdc48 affinity purified from budding yeast lysate in complex with the adaptor Shp1 in the act of unfolding substrate.

Visualization of the Cdc48 AAA+ ATPase protein unfolding pathway.

The Cdc48 AAA+ ATPase is an abundant and essential enzyme that unfolds substrates in multiple protein quality control pathways. The enzyme includes two conserved AAA+ ATPase cassettes, D1 and D2, that assemble as hexameric rings with D1 stacked above D2. Here, we report an ensemble of structures of Cdc48 affinity purified from lysate in complex with the adaptor Shp1 in the act of unfolding substrate.

Lysate-to-grid: Rapid Isolation of Native Complexes from Budding Yeast for Cryo-EM Imaging.

Single-particle electron cryo-microscopy (cryo-EM) is an effective tool to determine high-resolution structures of macromolecular complexes. Its lower requirements for sample concentration and purity make it an accessible method to determine structures of low-abundant protein complexes, such as those isolated from native sources. While there are many approaches to protein purification for cryo-EM, attaining suitable particle quality and abundance is generally the major bottleneck to the typical single-particle project workflow.

Active conformation of the p97-p47 unfoldase complex.

The p97 AAA+ATPase is an essential and abundant regulator of protein homeostasis that plays a central role in unfolding ubiquitylated substrates. Here we report two cryo-EM structures of human p97 in complex with its p47 adaptor. One of the conformations is six-fold symmetric, corresponds to previously reported structures of p97, and lacks bound substrate.

Structure of the Cdc48 segregase in the act of unfolding an authentic substrate.

The cellular machine Cdc48 functions in multiple biological pathways by segregating its protein substrates from a variety of stable environments such as organelles or multi-subunit complexes. Despite extensive studies, the mechanism of Cdc48 has remained obscure, and its reported structures are inconsistent with models of substrate translocation proposed for other AAA+ ATPases (adenosine triphosphatases). Here, we report a 3.