RESUMEN
Bacillus thuringiensis strains are well known for the production of insecticidal proteins upon sporulation and these proteins are deposited in parasporal crystalline inclusions. The majority of these insect-specific toxins exhibit three domains in the mature toxin sequence. However, other Cry toxins are structurally and evolutionarily unrelated to this three-domain family and little is known of their three dimensional structures, limiting our understanding of their mechanisms of action and our ability to engineer the proteins to enhance their function. Among the non-three domain Cry toxins, the Cry34Ab1 and Cry35Ab1 proteins from B. thuringiensis strain PS149B1 are required to act together to produce toxicity to the western corn rootworm (WCR) Diabrotica virgifera virgifera Le Conte via a pore forming mechanism of action. Cry34Ab1 is a protein of â¼14 kDa with features of the aegerolysin family (Pfam06355) of proteins that have known membrane disrupting activity, while Cry35Ab1 is a â¼44 kDa member of the toxin_10 family (Pfam05431) that includes other insecticidal proteins such as the binary toxin BinA/BinB. The Cry34Ab1/Cry35Ab1 proteins represent an important seed trait technology having been developed as insect resistance traits in commercialized corn hybrids for control of WCR. The structures of Cry34Ab1 and Cry35Ab1 have been elucidated to 2.15 Å and 1.80 Å resolution, respectively. The solution structures of the toxins were further studied by small angle X-ray scattering and native electrospray ion mobility mass spectrometry. We present here the first published structure from the aegerolysin protein domain family and the structural comparisons of Cry34Ab1 and Cry35Ab1 with other pore forming toxins.
Asunto(s)
Bacillus thuringiensis/química , Proteínas Bacterianas/química , Endotoxinas/química , Proteínas Fúngicas/química , Proteínas Hemolisinas/química , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Endotoxinas/genética , Proteínas Fúngicas/genética , Expresión Génica , Proteínas Hemolisinas/genética , Modelos Moleculares , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Pseudomonas fluorescens/genética , Pseudomonas fluorescens/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
The duplicated and the highly repetitive nature of the maize genome has historically impeded the development of true single nucleotide polymorphism (SNP) markers in this crop. Recent advances in genome complexity reduction methods coupled with sequencing-by-synthesis technologies permit the implementation of efficient genome-wide SNP discovery in maize. In this study, we have applied Complexity Reduction of Polymorphic Sequences technology (Keygene N.V., Wageningen, The Netherlands) for the identification of informative SNPs between two genetically distinct maize inbred lines of North and South American origins. This approach resulted in the discovery of 1,123 putative SNPs representing low and single copy loci. In silico and experimental (Illumina GoldenGate (GG) assay) validation of putative SNPs resulted in mapping of 604 markers, out of which 188 SNPs represented 43 haplotype blocks distributed across all ten chromosomes. We have determined and clearly stated a specific combination of stringent criteria (>0.3 minor allele frequency, >0.8 GenTrainScore and >0.5 Chi_test100 score) necessary for the identification of highly polymorphic and genetically stable SNP markers. Due to these criteria, we identified a subset of 120 high-quality SNP markers to leverage in GG assay-based marker-assisted selection projects. A total of 32 high-quality SNPs represented 21 haplotypes out of 43 identified in this study. The information on the selection criteria of highly polymorphic SNPs in a complex genome such as maize and the public availability of these SNP assays will be of great value for the maize molecular genetics and breeding community.
Asunto(s)
Mapeo Cromosómico , Cromosomas de las Plantas/genética , Marcadores Genéticos/genética , Genoma de Planta/genética , Polimorfismo de Nucleótido Simple/genética , Zea mays/genética , Cruzamiento , Cartilla de ADN , ADN de Plantas/genética , Ligamiento Genético , Genotipo , Reacción en Cadena de la PolimerasaRESUMEN
UNLABELLED: X-ray crystallography is the most widely used method to determine the 3D structure of protein molecules. One of the most difficult steps in protein crystallography is model-building, which consists of constructing a backbone and then amino acid side chains into an electron density map. Interpretation of electron density maps represents a major bottleneck in protein structure determination pipelines, and thus, automated techniques to interpret maps can greatly improve the throughput. We have developed WebTex, a simple and yet powerful web interface to TEXTAL, a program that automates this process of fitting atoms into electron density maps. TEXTAL can also be downloaded for local installation. AVAILABILITY: Web interface, downloadable binaries and documentation at http://textal.tamu.edu
Asunto(s)
Cristalografía/métodos , Internet , Modelos Químicos , Modelos Moleculares , Proteínas/química , Proteínas/ultraestructura , Programas Informáticos , Algoritmos , Simulación por Computador , Conformación Proteica , Análisis de Secuencia de Proteína/métodosRESUMEN
Non-crystallographic symmetry (NCS) averaging is a well known method for improving the quality of an electron-density map and thus aiding structure determination. Prior methods of NCS-operator determination based on estimated heavy-atom positions are prone to errors arising from inaccuracies in these coordinates or differences in the relative orientations of domains between molecules. In this paper, two real-space methods to determine NCS relationships from initial electron-density maps are presented. A brute-force method identifies matching regions in a map by local density correlation. A feature-based algorithm uses rotation-invariant features to reduce the computational time taken by the brute-force algorithm by filtering out regions that are likely to have dissimilar density patterns. This makes the feature-based algorithm faster and as accurate as the brute-force approach. Neither method requires the positions of heavy atoms or any information regarding the protein sequence. Both methods have been tested on a diverse range of experimentally phased maps and the correct NCS relationships were accurately identified for almost all of the test cases. The NCS operators obtained by the feature-based algorithm were used to perform NCS averaging and an improvement in map correlation was observed for some cases.
Asunto(s)
Biología Computacional/métodos , Algoritmos , Cristalografía por Rayos X , Electrones , Modelos Moleculares , Modelos Estadísticos , Conformación Molecular , Mycobacterium tuberculosis/enzimología , Reconocimiento de Normas Patrones Automatizadas , Fosfoglicerato-Deshidrogenasa/química , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas/química , Reproducibilidad de los ResultadosRESUMEN
A new software system called PHENIX (Python-based Hierarchical ENvironment for Integrated Xtallography) is being developed for the automation of crystallographic structure solution. This will provide the necessary algorithms to proceed from reduced intensity data to a refined molecular model, and facilitate structure solution for both the novice and expert crystallographer. Here, the features of PHENIXare reviewed and the recent advances in infrastructure and algorithms are briefly described.