Yi P.,Baylor College of Medicine |
Yi P.,Clayton Foundation for Research |
Wang Z.,National Center for Macromolecular Imaging |
Feng Q.,Baylor College of Medicine |
And 8 more authors.
Molecular Cell | Year: 2015
Estrogen receptor (ER/ESR1) is a transcription factor critical for development, reproduction, metabolism, and cancer. ER function hinges on its ability to recruit primary and secondary coactivators, yet structural information on the full-length receptor-coactivator complex to complement preexisting and sometimes controversial biochemical information is lacking. Here, we use cryoelectron microscopy (cryo-EM) to determine the quaternary structure of an active complex of DNA-bound ERα, steroid receptor coactivator 3 (SRC-3/NCOA3), and a secondary coactivator (p300/EP300). Our structural model suggests the following assembly mechanism for the complex: each of the two ligand-bound ERα monomers independently recruits one SRC-3 protein via the transactivation domain of ERα; the two SRC-3s in turn bind to different regions of one p300 protein through multiple contacts. We also present structural evidence for the location of activation function 1 (AF-1) in a full-length nuclear receptor, which supports a role for AF-1 in SRC-3 recruitment. © 2015 Elsevier Inc.
PubMed | University of California at San Diego, National Center for Macromolecular Imaging and Baylor College of Medicine
Type: | Journal: Journal of visualized experiments : JoVE | Year: 2014
Neurites, both dendrites and axons, are neuronal cellular processes that enable the conduction of electrical impulses between neurons. Defining the structure of neurites is critical to understanding how these processes move materials and signals that support synaptic communication. Electron microscopy (EM) has been traditionally used to assess the ultrastructural features within neurites; however, the exposure to organic solvent during dehydration and resin embedding can distort structures. An important unmet goal is the formulation of procedures that allow for structural evaluations not impacted by such artifacts. Here, we have established a detailed and reproducible protocol for growing and flash-freezing whole neurites of different primary neurons on electron microscopy grids followed by their examination with cryo-electron tomography (cryo-ET). This technique allows for 3-D visualization of frozen, hydrated neurites at nanometer resolution, facilitating assessment of their morphological differences. Our protocol yields an unprecedented view of dorsal root ganglion (DRG) neurites, and a visualization of hippocampal neurites in their near-native state. As such, these methods create a foundation for future studies on neurites of both normal neurons and those impacted by neurological disorders.