RESUMEN
In this study, we present a new way to predict the Zernike coefficients of optical system. We predict the Zernike coefficients through the function of image recognition in the neural network. It can reduce the mathematical operations commonly used in the interferometers and improve the measurement accuracy. We use the phase difference and the interference fringe as the input of the neural network to predict the coefficients respectively and compare the effects of the two models. In this study, python and optical simulation software are used to confirm the overall effect. As a result, all the Root-Mean-Square-Error (RMSE) are less than 0.09, which means that the interference fringes or the phase difference can be directly converted into coefficients. Not only can the calculation steps be reduced, but the overall efficiency can be improved and the calculation time reduced. For example, we could use it to check the performance of camera lenses.
RESUMEN
Vaccinia virus envelope protein A27 has multiple functions and is conserved in the Orthopoxvirus genus of the poxvirus family. A27 protein binds to cell surface heparan sulfate, provides an anchor for A26 protein packaging into mature virions, and is essential for egress of mature virus (MV) from infected cells. Here, we crystallized and determined the structure of a truncated form of A27 containing amino acids 21-84, C71/72A (tA27) at 2.2 Å resolution. tA27 protein uses the N-terminal region interface (NTR) to form an unexpected trimeric assembly as the basic unit, which contains two parallel α-helices and one unusual antiparallel α-helix; in a serpentine way, two trimers stack with each other to form a hexamer using the C-terminal region interface (CTR). Recombinant tA27 protein forms oligomers in a concentration-dependent manner in vitro in gel filtration. Analytical ultracentrifugation and multi-angle light scattering revealed that tA27 dimerized in solution and that Leu47, Leu51, and Leu54 at the NTR and Ile68, Asn75, and Leu82 at the CTR are responsible for tA27 self-assembly in vitro. Finally, we constructed recombinant vaccinia viruses expressing full length mutant A27 protein defective in either NTR, CTR, or both interactions; the results demonstrated that wild type A27 dimer/trimer formation was impaired in NTR and CTR mutant viruses, resulting in small plaques that are defective in MV egress. Furthermore, the ability of A27 protein to form disulfide-linked protein complexes with A26 protein was partially or completely interrupted by NTR and CTR mutations, resulting in mature virion progeny with increased plasma membrane fusion activity upon cell entry. Together, these results demonstrate that A27 protein trimer structure is critical for MV egress and membrane fusion modulation. Because A27 is a neutralizing target, structural information will aid the development of inhibitors to block A27 self-assembly or complex formation against vaccinia virus infection.
Asunto(s)
Proteínas Portadoras/química , Complejos Multiproteicos/química , Multimerización de Proteína , Virus Vaccinia/química , Proteínas Virales de Fusión/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X , Células HeLa , Humanos , Proteínas de la Membrana , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Virus Vaccinia/genética , Virus Vaccinia/metabolismo , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/metabolismoRESUMEN
Laminarinases hydrolyzing the ß-1,3-linkage of glucans play essential roles in microbial saccharide degradation. Here we report the crystal structures at 1.65-1.82 Å resolution of the catalytic domain of laminarinase from the thermophile Thermotoga maritima with various space groups in the ligand-free form or in the presence of inhibitors gluconolactone and cetyltrimethylammonium. Ligands were bound at the cleft of the active site near an enclosure formed by Trp-232 and a flexible GASIG loop. A closed configuration at the active site cleft was observed in some molecules. The loop flexibility in the enzyme may contribute to the regulation of endo- or exo-activity of the enzyme and a preference to release laminaritrioses in long chain carbohydrate hydrolysis. Glu-137 and Glu-132 are proposed to serve as the proton donor and nucleophile, respectively, in the retaining catalysis of hydrolyzation. Calcium ions in the crystallization media are found to accelerate crystal growth. Comparison of laminarinase and endoglucanase structures revealed the subtle difference of key residues in the active site for the selection of ß-1,3-glucan and ß-1,4-glucan substrates, respectively. Arg-85 may be pivotal to ß-1,3-glucan substrate selection. The similarity of the structures between the laminarinase catalytic domain and its carbohydrate-binding modules may have evolutionary relevance because of the similarities in their folds.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Celulasas/antagonistas & inhibidores , Celulasas/química , Inhibidores Enzimáticos/química , Thermotoga maritima/enzimología , beta-Glucanos/química , Calcio/química , Cristalografía por Rayos X , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología Estructural de ProteínaRESUMEN
NkBgl, a ß-glucosidase from Neotermes koshunensis, is a ß-retaining glycosyl hydrolase family 1 enzyme that cleaves ß-glucosidic linkages in disaccharide or glucose-substituted molecules. ß-Glucosidases have been widely used in several applications. For example, mutagenesis of the attacking nucleophile in ß-glucosidase has been conducted to convert it into a glycosynthase for the synthesis of oligosaccharides. Here, several high-resolution structures of wild-type or mutated NkBgl in complex with different ligand molecules are reported. In the wild-type NkBgl structures it was found that glucose-like glucosidase inhibitors bind to the glycone-binding pocket, allowing the buffer molecule HEPES to remain in the aglycone-binding pocket. In the crystal structures of NkBgl E193A, E193S and E193D mutants Glu193 not only acts as the catalytic acid/base but also plays an important role in controlling substrate entry and product release. Furthermore, in crystal structures of the NkBgl E193D mutant it was found that new glucoconjugates were generated by the conjugation of glucose (hydrolyzed product) and HEPES/EPPS/opipramol (buffer components). Based on the wild-type and E193D-mutant structures of NkBgl, the glucosidic bond of cellobiose or salicin was hydrolyzed and a new bond was subsequently formed between glucose and HEPES/EPPS/opipramol to generate new glucopyranosidic products through the transglycosylation reaction in the NkBgl E193D mutant. This finding highlights an innovative way to further improve ß-glucosidases for the enzymatic synthesis of oligosaccharides.
Asunto(s)
Glicoconjugados/metabolismo , Isópteros/enzimología , Oligosacáridos/metabolismo , beta-Glucosidasa/química , beta-Glucosidasa/metabolismo , Animales , Sitios de Unión , Cristalografía por Rayos X , Glucosa/metabolismo , Glicosilación , HEPES/metabolismo , Isópteros/química , Isópteros/genética , Isópteros/metabolismo , Modelos Moleculares , Mutación , Conformación Proteica , beta-Glucosidasa/genéticaRESUMEN
ß-glucosidases (EC 3.2.1.21) cleave ß-glucosidic linkages in disaccharide or glucose-substituted molecules and play important roles in fundamental biological processes. ß-Glucosidases have been widely used in agricultural, biotechnological, industrial and medical applications. In this study, a high yield expression (70-250 mg/l) in Escherichia coli of the three functional ß-glucosidase genes was obtained from the bacterium Clostridium cellulovorans (CcBglA), the fungus Trichoderma reesei (TrBgl2), and the termite Neotermes koshunensis (NkBgl) with the crystal structures of CcBglA, TrBgl2 and NkBgl, determined at 1.9Å, 1.63Å and 1.34Å resolution, respectively. The overall structures of these enzymes are similar to those belonging to the ß-retaining glycosyl hydrolase family 1, which have a classical (α/ß)(8)-TIM barrel fold. Each contains a slot-like active site cleft and a more variable outer opening, related to its function in processing different lengths of ß-1,4-linked glucose derivatives. The two essential glutamate residues for hydrolysis are spatially conserved in the active site. In both TrBgl2 and NkBgl structures, a Tris molecule was found to bind at the active site, explaining the slight inhibition of hydrolase activity observed in Tris buffer. Manganese ions at 10mM exerted an approximate 2-fold enzyme activity enhancement of all three ß-glucosidases, with CcBglA catalyzing the most efficiently in hydrolysis reaction and tolerating Tris as well as some metal inhibition. In summary, our results for the structural and functional properties of these three ß-glucosidases from various biological sources open important avenues of exploration for further practical applications.