Vitrification is the state-of-the-art specimen preparation technique for molecular electron microscopy (EM) and therefore negative staining may appear to be an outdated approach. In this paper we illustrate the specific advantages of negative staining, ensuring that this technique will remain an important tool for the study of biological macromolecules. Due to the higher image contrast, much smaller molecules can be visualized by negative staining. Also, while molecules prepared by vitrification usually adopt random orientations in the amorphous ice layer, negative staining tends to induce preferred orientations of the molecules on the carbon support film. Combining negative staining with image classification techniques makes it possible to work with very heterogeneous molecule populations, which are difficult or even impossible to analyze using vitrified specimens.

Ubiquitin-like proteins (UBLs) are conjugated by dynamic E1-E2-E3 enzyme cascades. E1 enzymes activate UBLs by catalysing UBL carboxy-terminal adenylation, forming a covalent E1 throught UBL thioester intermediate, and generating a thioester-linked E2 throught UBL product, which must be released for subsequent reactions. Here we report the structural analysis of a trapped UBL activation complex for the human NEDD8 pathway, containing NEDD8's heterodimeric E1 (APPBP1-UBA3), two NEDD8s (one thioester-linked to E1, one noncovalently associated for adenylation), a catalytically inactive E2 (Ubc12), and MgATP. The results suggest that a thioester switch toggles E1-E2 affinities. Two E2 binding sites depend on NEDD8 being thioester-linked to E1. One is unmasked by a striking E1 conformational change. The other comes directly from the thioester-bound NEDD8. After NEDD8 transfer to E2, reversion to an alternate E1 conformation would facilitate release of the E2 throught NEDD8 thioester product. Thus, transferring the UBL's thioester linkage between successive conjugation enzymes can induce conformational changes and alter interaction networks to drive consecutive steps in UBL cascades.

The structure of the U-box in the essential Saccharomyces cerevisiae pre-mRNA splicing factor Prp19p has been determined by NMR. The conserved zinc-binding sites supporting the crossbrace arrangement in RING-finger domains are replaced by hydrogen-bonding networks in the Ubox. These hydrogen-bonding networks are necessary for the structural stabilization and activity of the U-box. A conservative Val→Ile point mutation in the Prp19p U-box domain leads to premRNA splicing defects in vivo. NMR analysis of this mutant shows that the substitution disrupts structural integrity of the U-box domain. Furthermore, comparison of the Prp19p U-box domain with known RING-E2 complex structures demonstrates that both U-box and RING-fingers contain a conserved interaction surface. Mutagenesis of residues at this interface, while not perturbing the structure of the U-box, abrogates Prp19p function in vivo. These comparative structural and functional analyses imply that the U-box and its associated ubiquitin ligase activity are critical for Prp19p function in vivo.Ubiquitin (Ub) targeting of proteins for degradation by the proteosome involves polyubiquination of substrate proteins via an enzyme cascade consisting of activating (E1), conjugating (E2) and ligating (E3) enzymes. E3 ubiquitin ligases vary widely in size, composition and enzymology, reflecting their regulatory role in substrate recognition 1 . HECT and RING finger domain-containing proteins constitute two classes of E3 ligases. HECT domains bind Ub through a thioester bond and transfer Ub directly to substrate. RING finger E3s facilitate the transfer of Ub from the E2 to the substrate, rather than binding Ub directly.Correspondence should be addressed to K.L.G. kathy.gould@mcmail.vanderbilt.edu or W.J.C. walter.chazin@vanderbilt.edu. 3 These authors contributed equally to this work. Competing interests statementThe authors declare that they have no competing financial interests. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptA third class of E3 ubiquitin ligases has been recently identified 2,3 . This class of proteins contains a U-box motif, first identified in Saccharomyces cerevisiae Ufd2p. Ufd2p promotes the elongation of poly-ubiquitin chains in a U-box-dependent manner (recently termed an 'E4' activity) 4 . An alignment of U-boxes and RING motifs indicated that U-boxes lack the strictly conserved histidine and cysteine Zn 2+ -chelating residues found in RING fingers, but they share a similar pattern of hydrophobic and polar amino acids (Fig. 1a), raising the possibility that they have similar folds 5 .S. cerevisiae Prp19p is an essential pre-mRNA splicing factor that contains an N-terminal Ubox 6,7 . Interestingly, a mutation within the Prp19p U-box that results in the substitution of an isoleucine for a conserved valine (prp19-1) leads to pre-mRNA splicing defects and the disruption of several key protein-protein interactions within the spliceosome 8,9 . The profound physiological consequences that result from th...