RESUMEN
CryoEM in structural biology is currently served by three public archives-EMDB for 3DEM reconstructions, PDB for models built from 3DEM reconstructions, and EMPIAR for the raw 2D image data used to obtain the 3DEM reconstructions. These archives play a vital role for both the structural community and the wider biological community in making the data accessible so that results may be reused, reassessed, and integrated with other structural and bioinformatics resources. The important role of the archives is underpinned by the fact that many journals mandate the deposition of data to PDB and EMDB on publication. The field is currently undergoing transformative changes where on the one hand high-resolution structures are becoming a routine occurrence while on the other hand electron tomography is enabling the study of macromolecules in the cellular context. Concomitantly the archives are evolving to best serve their stakeholder communities. In this chapter, we describe the current state of the archives, resources available for depositing, accessing, searching, visualizing and validating data, on-going community-wide initiatives and opportunities, and challenges for the future.
Asunto(s)
Microscopía por Crioelectrón/estadística & datos numéricos , Bases de Datos de Proteínas/provisión & distribución , Tomografía con Microscopio Electrónico/estadística & datos numéricos , Proteínas/ultraestructura , Programas Informáticos , Biología Computacional/estadística & datos numéricos , Microscopía por Crioelectrón/métodos , Bases de Datos como Asunto , Tomografía con Microscopio Electrónico/métodos , Difusión de la Información , Modelos MolecularesRESUMEN
A 22-year-old man was hospitalized after unexplained seizure-like activity and unresponsiveness. A urine toxicology screen was negative for salicylates, acetaminophen, alcohol, and drugs of abuse. Medical history was insignificant with the exception of recent (within 2 wks) ingestion of Hydroxycut is a dietary supplement purported to be energy enhancing, muscle building, and fat burning. The agent contains ephedra alkaloids and caffeine, which are both central nervous system stimulants; the etiology of seizure was attributed to their consumption. Due to a significant number of reported adverse events, the United States Food and Drug Administration (FDA) proposed regulations for dietary supplements containing ephedra alkaloids and requested an independent review of case reports linked to these products. Because herbal products are not subject to the same rigorous FDA regulations required for prescription and over-the-counter products, consumers unknowingly risk adverse effects when taking these products. Questioning patients about consumption of herbal products should be part of routine medical visits.
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Estimulantes del Sistema Nervioso Central/efectos adversos , Citratos/efectos adversos , Ephedra sinica , Convulsiones/inducido químicamente , Adulto , Cafeína/efectos adversos , Combinación de Medicamentos , Medicamentos Herbarios Chinos/efectos adversos , Efedrina/efectos adversos , Humanos , Masculino , Fitoterapia , Ácidos Picolínicos/efectos adversos , Preparaciones de Plantas , Polisacáridos/efectos adversos , Convulsiones/psicología , Teobromina/efectos adversos , Teofilina/efectos adversosRESUMEN
Outer surface protein C (OspC) is a major antigen on the surface of the Lyme disease spirochete, Borrelia burgdorferi, when it is being transmitted to humans. Crystal structures of OspC have been determined for strains HB19 and B31 to 1.8 and 2.5 A resolution, respectively. The three-dimensional structure is predominantly helical. This is in contrast to the structure of OspA, a major surface protein mainly present when spirochetes are residing in the midgut of unfed ticks, which is mostly beta-sheet. The surface of OspC that would project away from the spirochete's membrane has a region of strong negative electrostatic potential which may be involved in binding to positively charged host ligands. This feature is present only on OspCs from strains known to cause invasive human disease.
Asunto(s)
Antígenos Bacterianos/química , Proteínas de la Membrana Bacteriana Externa/química , Grupo Borrelia Burgdorferi/química , Borrelia burgdorferi , Enfermedad de Lyme/microbiología , Secuencia de Aminoácidos , Antígenos Bacterianos/genética , Proteínas de la Membrana Bacteriana Externa/genética , Sitios de Unión , Grupo Borrelia Burgdorferi/genética , Grupo Borrelia Burgdorferi/patogenicidad , Cristalografía por Rayos X , Dimerización , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes , Alineación de Secuencia , Eliminación de Secuencia , Electricidad EstáticaRESUMEN
Vsp surface lipoproteins are serotype-defining antigens of relapsing fever spirochetes that undergo multiphasic antigenic variation to avoid the immune response. One of these proteins, VspA of Borrelia turicatae, is also associated with neurotropism in infected mice. Vsp proteins are highly polymorphic in sequence, which may relate to their specific antibody reactivities and host cell interactions. To determine whether sequence variations affect protein structure, we compared B. turicatae VspA with three related proteins: VspB of B. turicatae, Vsp26 of the relapsing fever agent Borrelia hermsii, and OspC of the Lyme disease spirochete Borrelia burgdorferi. Recombinant non-lipidated proteins were purified by affinity or ion exchange chromatography. Circular dichroism spectra revealed similar, highly alpha-helical secondary structures for all four proteins. In vitro assays demonstrated protease-resistant, thermostable Vsp cores starting at a conserved serine at position 34 (Ser(34)). All proteins aggregate as dimers in solution. In situ trypsin treatment and surface protein cross-linking showed that the native lipoproteins also form protease-resistant dimers. These findings indicate that Vsp proteins have a common compact fold and that their established functions are based on localized polymorphisms. Two forms of VspA crystals suitable for structure determination by x-ray diffraction methods have been obtained.
Asunto(s)
Antígenos Bacterianos/química , Proteínas de la Membrana Bacteriana Externa/química , Borrelia/química , Lipoproteínas/química , Secuencia de Aminoácidos , Antígenos Bacterianos/aislamiento & purificación , Antígenos Bacterianos/metabolismo , Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Proteínas de la Membrana Bacteriana Externa/metabolismo , Western Blotting , Borrelia/clasificación , Borrelia/inmunología , Cromatografía en Gel , Dicroismo Circular , Cristalización , Cristalografía por Rayos X , Dimerización , Endopeptidasas/metabolismo , Evolución Molecular , Lipoproteínas/aislamiento & purificación , Lipoproteínas/metabolismo , Espectrometría de Masas , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Análisis de Secuencia de Proteína , SolucionesRESUMEN
Pseudorandom encoding (PRE) is a statistics-based procedure in which a pure-phase spatial light modulator (SLM) can yield, on the average, the prescribed diffraction pattern specified by the user. We seek to combine PRE with the optimization of an aperture-based target function. The target function is a fully complex input transmittance, unrealizable by a phase-only SLM, that generates a prescribed light intensity. The optimization is done to increase the diffraction efficiency of the overall process. We compare three optimization methods-Monte Carlo simulation, a genetic algorithm, and a gradient search-for maximizing the diffraction efficiency of a spot-array generator. Calculated solutions are then encoded by PRE, and the resulting diffraction patterns are computer simulated. Details on the complexity of each procedure are furnished, as well as comparisons on the quality, such as uniformity of the output spot array.
Asunto(s)
Luz , Modelos Teóricos , Simulación por Computador , Humanos , Método de Montecarlo , Dispersión de RadiaciónRESUMEN
Outer surface protein A (OspA) is a major lipoprotein of the Borrelia burgdorferi spirochete, the causative agent of Lyme disease. Vaccination with OspA generates an immune response that can prevent bacterial transmission to a mammalian host during the attachment of an infected tick. However, the protective capacity of immune sera cannot be predicted by measuring total anti-OspA antibody. The murine monoclonal antibody LA-2 defines an important protective B-cell epitope of OspA against which protective sera have strong levels of reactivity. We have now mapped the LA-2 epitope of OspA using both NMR chemical-shift perturbation measurements in solution and X-ray crystal structure determination. LA-2 recognizes the three surface-exposed loops of the C-terminal domain of OspA that are on the tip of the elongated molecule most distant from the lipid-modified N terminus. The structure suggests that the natural variation at OspA sequence position 208 in the first loop is a major limiting factor for antibody cross-reactivity between different Lyme disease-causing Borrelia strains. The unusual Fab-dominated lattice of the crystal also permits a rare view of antigen flexibility within an antigen:antibody complex. These results provide a rationale for improvements in OspA-based vaccines and suggest possible designs for more direct tests of antibody protective levels in vaccinated individuals.
Asunto(s)
Antígenos de Superficie/química , Antígenos de Superficie/inmunología , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/inmunología , Grupo Borrelia Burgdorferi/inmunología , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Lipoproteínas , Vacunas contra Enfermedad de Lyme/química , Vacunas contra Enfermedad de Lyme/inmunología , Enfermedad de Lyme/inmunología , Secuencia de Aminoácidos , Complejo Antígeno-Anticuerpo/química , Complejo Antígeno-Anticuerpo/inmunología , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Antígenos de Superficie/genética , Proteínas de la Membrana Bacteriana Externa/genética , Vacunas Bacterianas , Grupo Borrelia Burgdorferi/química , Grupo Borrelia Burgdorferi/genética , Cristalografía por Rayos X , Mapeo Epitopo , Epítopos de Linfocito B/genética , Variación Genética/genética , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Vacunas contra Enfermedad de Lyme/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
Two crystal forms of recombinant p26 capsid protein (CA) from the equine infectious anemia virus (EIAV) have in common an antiparallel four-helix bundle dimer interface between N-terminal domains (NTDs). The dimer interface provides a lenient scaffold to accommodate the wide sequence variation in these helices within lentivirus CA. Pairs of dimers weakly associate to form exact or approximate D2 symmetry tetramers. In one of the two crystal forms, the tetramers are linked via dimerization of C-terminal domains (CTDs). We propose that the observed NTD and CTD homodimer interactions are involved in the assembly of the lentivirus capsid. The NTD homodimer shape readily suggests a model for the mature capsid core, based on hexagonal packing with dimensions and surface topology resembling described EIAV capsid cores. Combining available data for human immunodeficiency virus and EIAV CA, we also propose an assembly pathway for maturation of the lentivirus capsid core following proteolytic cleavage of the gag polyprotein precursor.
Asunto(s)
Cápside/biosíntesis , Virus de la Anemia Infecciosa Equina/fisiología , Proteínas del Núcleo Viral/biosíntesis , Secuencia de Aminoácidos , Cápside/química , Cristalografía por Rayos X , Dimerización , VIH/fisiología , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Proteínas del Núcleo Viral/química , Ensamble de VirusRESUMEN
OspA (outer surface protein A) is an abundant immunogenic lipoprotein of the Lyme disease spirochete Borrelia burgdorferi. The crystal structure of a soluble recombinant form of OspA was solved in a complex with the Fab fragment of mouse monoclonal antibody 184.1 and refined to a resolution of 1.9 A. OspA has a repetitive antiparallel beta topology with an unusual nonglobular region of "freestanding" sheet connecting globular N- and C-terminal domains. Arrays of residues with alternating charges are a predominant feature of the folding pattern in the nonglobular region. The 184.1 epitope overlaps with a well conserved surface in the N-terminal domain, and a hydrophobic cavity buried in a positively charged cleft in the C-terminal domain is a potential binding site for an unknown ligand. An exposed variable region on the C-terminal domain of OspA is predicted to be an important factor in the worldwide effectiveness of OspA-based vaccines.
Asunto(s)
Antígenos de Superficie/química , Proteínas de la Membrana Bacteriana Externa/química , Grupo Borrelia Burgdorferi/química , Lipoproteínas , Enfermedad de Lyme/microbiología , Pliegue de Proteína , Secuencia de Aminoácidos , Animales , Antígenos de Superficie/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas , Grupo Borrelia Burgdorferi/inmunología , Cristalización , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Ratones , Datos de Secuencia Molecular , Unión ProteicaRESUMEN
The Borrelia burgdorferi outer surface lipoprotein OspA is a current focus for vaccine development to prevent Lyme disease infection. A soluble, recombinant form of the protein lacking the amino-terminal lipid membrane anchor was cocrystallized with the Fab fragment of an agglutinating mouse monoclonal antibody. The crystals belong to space group P2(1)2(1)2(1), with a = 90.0 A, b = 91.9 A, and c = 102.9 A and they were found to diffract to a maximum resolution of 2.8 A using synchrotron radiation.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Complejo Antígeno-Anticuerpo/química , Antígenos Bacterianos/ultraestructura , Antígenos de Superficie/química , Proteínas de la Membrana Bacteriana Externa/química , Grupo Borrelia Burgdorferi/química , Cristalografía por Rayos X , Fragmentos Fab de Inmunoglobulinas/inmunología , Lipoproteínas , Animales , Antígenos Bacterianos/inmunología , Antígenos de Superficie/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas , Grupo Borrelia Burgdorferi/inmunología , Cristalización , Ratones , SolubilidadRESUMEN
A simple modification to the fast T(1) translation function of Crowther & Blow [Crowther & Blow (1967). Acta Cryst. 23, 544-548] can reduce systematic error in cases where the input set of oriented search models represents a fraction of the scattering matter of the unknown crystal unit cell. The observed Patterson function is modified by a partial self-vector subtraction (SVS) and then the residual Patterson origin is removed. In a test case where a protein/DNA complex crystal was searched using a protein-only model, the highest signal- to-noise ratios for translation-search maps were obtained either with origin removal alone or partial SVS combined with origin removal. Origin removal is likely to be a generally useful alternative to SVS for the function.
RESUMEN
Tyrosine 195 is located in the center of the active site cleft of cyclodextrin glycosyltransferase (EC 2.4.1.19) from Bacillus circulans strain 251. Alignment of amino acid sequences of CGTases and alpha-amylases, and the analysis of the binding mode of the substrate analogue acarbose in the active site cleft [Strokopytov, B., et al. (1995) Biochemistry 34, (in press)], suggested that Tyr195 plays an important role in cyclization of oligosaccharides. Tyr195 therefore was replaced with Phe (Y195F), Trp (Y195W), Leu (Y195L), and Gly (Y195G). Mutant proteins were purified and crystallized, and their X-ray structures were determined at 2.5-2.6 angstrum resolution, allowing a detailed comparison of their biochemical properties and three-dimensional structures with those of the wild-type CGTase protein. The mutant proteins possessed significantly reduced cyclodextrin forming and coupling activities but were not negatively affected in the disproportionation and saccharifying reactions. Also under production process conditions, after a 45 h incubation with a 10% starch solution, the Y195W, Y195L, and Y195G mutants showed a lower overall conversion of starch into cyclodextrins. These mutants produced a considerable amount of linear maltooligosaccharides. The presence of aromatic amino acids (Tyr or Phe) at the Tyr195 position thus appears to be of crucial importance for an efficient cyclization reaction, virtually preventing the formation of linear products. Mass spectrometry of the Y195L reaction mixture, but not that of the other mutants and the wild type, revealed a shift toward the synthesis (in low yields) of larger products, especially of beta- and gamma- (but no alpha-) cyclodextrins and minor amounts of delta-, epsilon-, zeta- and eta-cyclodextrins.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Bacillus/enzimología , Bacillus/genética , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Mutación , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión/genética , Cristalografía por Rayos X , ADN Bacteriano/genética , Escherichia coli/genética , Glucosiltransferasas/química , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Mutagénesis Sitio-Dirigida , Plásmidos/genética , Conformación Proteica , Homología de Secuencia de Aminoácido , Tirosina/genética , alfa-Amilasas/genéticaRESUMEN
OBJECTIVE: To develop a clinical decision model to compare the outcome of therapy with digoxin versus diltiazem for short-term control of ventricular response rate (VRR) in patients with atrial fibrillation or atrial flutter. DESIGN: Review of data from two studies that examined the percentages of response and frequency of adverse reactions in patients treated with intravenous digoxin or diltiazem to control VRR in atrial fibrillation or flutter. We constructed a clinical decision model and performed sensitivity analysis to determine if the model's predictions could be altered. SETTING: Large teaching, university hospitals. PARTICIPANTS: Adults age 18 years or older treated with intravenous digoxin or intravenous diltiazem for atrial fibrillation or flutter (VRR > or = 120 beats/min). Patients with severe heart failure New York Heart Association class III or IV, a surgical procedure prior to the exacerbation, or an acute myocardial infarction were excluded. MEASUREMENTS AND MAIN RESULTS: We measured VRR control after 1 and 24 hours of therapy (VRR < 100 beats/min or decrease of > or = 20%) and assessed the likelihood that a patient would suffer an adverse drug reaction. Initial assumptions were that the probability digoxin would achieve VRR control was 0.10 (95% confidence interval 0.04-0.20) at 1 hour and 0.70 (95% CI 0.56-0.80) at 24 hours; the probability that diltiazem would achieve VRR control was 0.94 (95% CI 0.82-0.99) at 1 hour and 0.83 (95% CI 0.68-0.94) at 24 hours; and the probability of no serious adverse drug reaction would be 0.90 (95% CI 0.80-0.96) for digoxin and 0.96 (95% CI 0.86-0.98) for diltiazem. RESULTS: Diltiazem was superior to digoxin with respect to the composite end point score at 1 hour (91.20 vs 17.29) and 24 hours (81.65 vs 66.43). Digoxin was superior to diltiazem at 24 hours only if the VRR was assumed to be at the highest 95% CI limit for digoxin and simultaneously at the lowest 95% CI for diltiazem (74.62 vs 68.63). CONCLUSIONS: Clinical decision analysis suggests that intravenous diltiazem is superior to intravenous digoxin in controlling VRR in patients with atrial fibrillation or flutter.
Asunto(s)
Fibrilación Atrial/tratamiento farmacológico , Aleteo Atrial/tratamiento farmacológico , Técnicas de Apoyo para la Decisión , Digoxina/uso terapéutico , Diltiazem/uso terapéutico , Función Ventricular/efectos de los fármacos , Adolescente , Adulto , Digoxina/administración & dosificación , Digoxina/farmacología , Diltiazem/administración & dosificación , Diltiazem/farmacología , Femenino , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Infusiones Intravenosas , Masculino , Sensibilidad y Especificidad , Función Ventricular/fisiologíaRESUMEN
Tryptophan biosynthesis in Escherichia coli is regulated by the product of the trpR gene, the tryptophan (Trp) repressor. Trp aporepressor binds the corepressor, L-tryptophan, to form a holorepressor complex, which binds trp operator DNA tightly, and inhibits transcription of the tryptophan biosynthetic operon. The conservation of trp operator sequences among enteric Gram-negative bacteria suggests that trpR genes from other bacterial species can be cloned by complementation in E. coli. To clone trpR homologues, a deletion of the E. coli trpR gene, delta trpR504, was made on a plasmid by site-directed mutagenesis, then crossed onto the E. coli genome. Plasmid clones of the trpR genes of Enterobacter aerogenes and Enterobacter cloacae were isolated by complementation of the delta trpR504 allele, scored as the ability to repress beta-galactosidase synthesis from a prophage-borne trpE-lacZ gene fusion. The predicted amino acid sequences of four enteric TrpR proteins show differences, clustered on the backside of the folded repressor, opposite the DNA-binding helix-turn-helix substructures. These differences are predicted to have little effect on the interactions of the aporepressor with tryptophan, holorepressor with operator DNA, or tandemly bound holorepressor dimers with one another. Although there is some variation observed at the dimer interface, interactions predicted to stabilize the interface are conserved. The phylogenetic relationships revealed by the TrpR amino acid sequence alignment agree with the results of others.
Asunto(s)
Proteínas Bacterianas , Secuencia Conservada , Enterobacteriaceae/genética , Genes Bacterianos/genética , Proteínas Represoras/genética , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , ADN Bacteriano/análisis , Enterobacter/genética , Escherichia coli/genética , Prueba de Complementación Genética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Operón/genética , Filogenia , Conformación Proteica , Secuencias Reguladoras de Ácidos Nucleicos , Salmonella typhimurium/genética , Alineación de Secuencia , Eliminación de Secuencia/genéticaRESUMEN
The cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) gene from Bacillus circulans strain 251 was cloned and sequenced. It was found to code for a mature protein of 686 amino acid residues, showing 75% identity to the CGTase from B. circulans strain 8. The X-ray structure of the CGTase was elucidated in a maltodextrin-dependent crystal form and refined against X-ray diffraction data to 2.0 A resolution. The structure of the enzyme is nearly identical to the CGTase from B. circulans strain 8. Three maltose binding sites are observed at the protein surface, two in domain E and one in domain C. The maltose-dependence of CGTase crystallization can be ascribed to the proximity of two of the maltose binding sites to intermolecular crystal contacts. The maltose molecules bound in the E domain interact with several residues implicated in a raw starch binding motif conserved among a diverse group of starch converting enzymes.
Asunto(s)
Bacillus/enzimología , Glucosiltransferasas/genética , Maltosa/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Secuencia de Carbohidratos , Clonación Molecular , Gráficos por Computador , ADN Bacteriano , Glucosiltransferasas/química , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Difracción de Rayos XRESUMEN
The crystal structure of trp repressor tandemly bound in a 2:1 complex to a 16-base-pair palindromic DNA containing a central trp operator half-site has been determined and refined to 2.4 A resolution. Despite dramatically different DNA sequence contexts and crystallization conditions, the protein/DNA interface is essentially identical to that seen in the original trp repressor/operator complex structure. Water-mediated sequence recognition by trp repressor is likely to be related to the unusual end-on approach of the recognition helix (E), which allows sharing of the major groove by tandem dimers. The tandem complex model accounts for the mutational sensitivity of all trp operator base pairs. The structure also provides the first detailed view of the tandem interaction, revealing a key role for the amino-terminal arms.
Asunto(s)
Proteínas Bacterianas , Regiones Operadoras Genéticas , Proteínas Represoras/metabolismo , Secuencia de Bases , Gráficos por Computador , Cristalografía por Rayos X , ADN Bacteriano/química , ADN Bacteriano/metabolismo , Escherichia coli/genética , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Proteínas Represoras/químicaRESUMEN
Cocrystals of a 2:1 complex of trp repressor dimers with a DNA duplex containing a single, central operator half-site sequence are described. Crystals with different morphologies grew under diverse crystallization conditions within days to weeks by hanging-drop vapor-diffusion. Twinned rods split along their longitudinal cleft produce single crystals with space group C2 and unit cell dimensions a = 112.34 A, b = 90.16 A, c = 58.65 A and beta = 113.92 degrees. The crystals diffract to 2.4 A and are thus suitable for structural analysis by X-ray diffraction. Several heavy-atom derivative cocrystals have been obtained with iodouridine-substituted DNAs.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de Unión al ADN/química , Escherichia coli/genética , Regiones Operadoras Genéticas , Proteínas Represoras/química , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Proteínas de Unión al ADN/metabolismo , Datos de Secuencia Molecular , Proteínas Represoras/metabolismoRESUMEN
Crystals of cyclomaltodextrin glucanotransferase from Bacillus circulans (EC 2.4.1.19) suitable for high-resolution X-ray analysis were obtained by vapor diffusion against 60% (v/v) 2-methyl 2,4-pentanediol buffered with 100 mM-sodium Hepes, pH 7.55. The crystals have P2(1)2(1)2(1) space group symmetry, with a = 120.4 A, b = 110.9 A and c = 66.4 A, and contain one molecule of 68,000 in the asymmetric unit. Growth of single enzyme crystals was found to require the presence of either alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, or maltose in high molar excess, a requirement that could not be fulfilled by glucose, the basic building block of these compounds. Although the exact role of cyclic and linear maltodextrins in enzyme crystallization is not yet known, we have preliminary evidence that these compounds are degraded by the enzyme in the crystallization droplet.
Asunto(s)
Bacillus/enzimología , Glucosiltransferasas/metabolismo , Polisacáridos/metabolismo , Cristalización , Glucosiltransferasas/aislamiento & purificación , Peso Molecular , Unión Proteica , Conformación Proteica , Difracción de Rayos XRESUMEN
Alcohol oxidase, purified from the yeast Hansenula polymorpha, was crystallized in vitro for the purpose of determining its structure at atomic resolution by X-ray diffraction methods. The crystals obtained yielded only extremely weak diffraction patterns: the maximal resolution observed was in the best case 6 A. Electron microscopy of thin sections indicated that most crystals showed lattice defects which might explain the poor diffraction patterns: most surprising was the appearance of large holes interrupting an otherwise regular lattice in one of the crystal forms examined. Our results indicate that transmission electron microscopy is a suitable tool for the inspection of crystals to be used in X-ray crystallography. The method allows rapid determination of lattice defects and enables optimization of crystallization conditions.
Asunto(s)
Oxidorreductasas de Alcohol , Pichia/enzimología , Saccharomycetales/enzimología , Cloruro de Calcio , Cristalización , Ditiotreitol , Ácido Edético , Sustancias Macromoleculares , Microscopía Electrónica , Peso Molecular , Difracción de Rayos XRESUMEN
The crystal structure of the trp repressor/operator complex shows an extensive contact surface, including 24 direct and 6 solvent-mediated hydrogen bonds to the phosphate groups of the DNA. There are no direct hydrogen bonds or non-polar contacts to the bases that can explain the repressor's specificity for the operator sequence. Rather, the sequence seems to be recognized indirectly through its effects on the geometry of the phosphate backbone, which in turn permits the formation of a stable interface. Water-mediated polar contacts to the bases also appear to contribute part of the specificity.