Published in Methods in Enzymology
The SARS-CoV-2 3a protein is a putative ion channel implicated in virus life cycle and pathogenesis. We recently expressed, purified, and reconstituted 3a into lipid nanodiscs to solve its structure by cryo-EM to 2.1 Å resolution. In this chapter, we describe methods we developed in order to facilitate the study of this protein in other laboratorie...
Published in Methods in Enzymology
The actin cytoskeleton is very dynamic and highly regulated by multiple associated proteins in vivo. Understanding how this system of proteins functions in the processes of actin network assembly and disassembly requires methods to dissect the mechanisms of activity of individual factors and of multiple factors acting in concert. The advent of sing...
Published in Methods in Enzymology
The immunocapture-based ELISA for extracellular vesicles (EVs)/exosomes, originally described in 2009 by Logozzi and colleagues, allows to capture, detect, characterize and quantify extracellular vesicles in both human body fluids and cell culture supernatants. It is based on the use of two antibodies directed one against a typical exosomal houseke...
Published in Methods in Enzymology
Electron crystallography of 2D protein crystals can determine the structure of membrane embedded proteins at high resolution. Images or electron diffraction patterns are recorded with the electron microscope of the frozen hydrated samples, and the 3D structure of the proteins is then determined by computer data processing. Here we introduce the ima...
Published in Methods in Enzymology
Electron crystallography is a powerful technique for the structure determination of membrane proteins as well as soluble proteins. Sample preparation for 2D membrane protein crystals is a crucial step, as proteins have to be prepared for electron microscopy at close to native conditions. In this review, we discuss the factors of sample preparation ...
Published in Methods in Enzymology
Ubiquitin signaling requires tight control of all aspects of protein ubiquitination, including the timing, locale, extent, and type of modification. Dysregulation of any of these signaling features can lead to severe human disease. One key mode of regulation is through the controlled removal of the ubiquitin signal by dedicated families of protease...
Published in Methods in Enzymology
Protein (poly-)ubiquitination is a posttranslational modification that plays a key role in almost all cellular processes. It involves the installment of either single ubiquitin (Ub) moieties or one of eight different polyUb linkage types, each giving a distinct cellular outcome. Deubiquitinating enzymes (DUBs) reverse Ub signaling by disassembly of...
Published in Methods in Enzymology
This chapter discusses strategies for designing selective excitation (or inversion) pulses, and the applications of such pulses to multidimensional experiments for assignment of resonances, derivation of coupling constants, and determination of relaxation rates. The development of sophisticated pulses for selective excitation and inversion has trig...
Published in Methods in Enzymology
The ability of white and brown adipose tissue to efficiently take up long-chain fatty acids is key to their physiological functions in energy storage and thermogenesis, respectively. Several approaches have been taken to determine uptake rates by cultured cells and primary adipocytes including radio- and fluorescently labeled fatty acids. In additi...
Published in Methods in Enzymology
