PubMed Health⌕ Search

Biomedical subjects

J Badger

Publications and source records attributed to J Badger.

At least 19 recordsLinked to original sources
PubMed2005

Structural analysis of a set of proteins resulting from a bacterial genomics project.

The targets of the Structural GenomiX (SGX) bacterial genomics project were proteins conserved in multiple prokaryotic organisms with no obvious sequence homolog in the Protein Data Bank of known structures. The outcome of this work was 80 structures, covering 60 unique sequences and 49 different genes. Experimental phase determination from proteins incorporating Se-Met was carried out for 45 structures with most of the remainder solved by molecular replacement using members of the experimentally phased set as search models. An automated tool was developed to deposit these structures in the Protein Data Bank, along with the associated X-ray diffraction data (including refined experimental phases) and experimentally confirmed sequences. BLAST comparisons of the SGX structures with structures that had appeared in the Protein Data Bank over the intervening 3.5 years since the SGX target list had been compiled identified homologs for 49 of the 60 unique sequences represented by the SGX structures. This result indicates that, for bacterial structures that are relatively easy to express, purify, and crystallize, the structural coverage of gene space is proceeding rapidly. More distant sequence-structure relationships between the SGX and PDB structures were investigated using PDB-BLAST and Combinatorial Extension (CE). Only one structure, SufD, has a truly unique topology compared to all folds in the PDB.

Databases, Protein↗
PubMed2005

Deposit3D: a tool for automating structure depositions to the Protein Data Bank.

Almost all successful protein structure-determination projects in the public sector culminate in a structure deposition to the Protein Data Bank (PDB). In order to expedite the deposition process, Deposit3D has been developed. This command-line script calculates or gathers all the required structure-deposition information and outputs this data into a mmCIF file for subsequent upload through the RCSB PDB ADIT interface. Deposit3D might be particularly useful for structural genomics pipeline projects because it allows workers involved with various stages of a structure-determination project to pool their different categories of annotation information before starting a deposition session.

Automation↗
PubMed2001

Bacterial invasion and transcytosis in transfected human brain microvascular endothelial cells.

Most cases of neonatal bacterial meningitis develop as a result of a hematogenous spread, but it is not clear how circulating bacteria cross the blood-brain barrier. Attempts to answer these questions have been hampered by the lack of a reliable model of the human blood-brain barrier. Human brain microvascular endothelial cells (HBMEC) were isolated and transfected with a pBR322 based plasmid containing simian virus 40 large T antigen (SV40-LT). The transfected HBMEC exhibited similar brain endothelial cell characteristics as the primary HBMEC, i.e. gamma glutamyl transpeptidase and a high transendothelial electrical resistance. Escherischia coli and Citrobacter spp, two important Gram-negative bacilli causing neonatal meningitis, were found to transcytose across primary and transfected HBMEC, without affecting the integrity of the monolayer. In addition, E. coli and C. freundii invaded transfected HBMEC as shown previously with primary HBMEC. We conclude that E. coli and C. freundii are able to invade and transcytose HBMEC and these bacterial-HBMEC interactions are similar between primary and transfected HBMEC. Therefore, our transfected HBMEC should be useful for studying pathogenesis of CNS infections.

Blood-Brain Barrier↗
PubMed2001

Analysis of codon usage patterns of bacterial genomes using the self-organizing map.

Codon usage varies both between organisms and between different genes in the same organism. This observation has been used as a basis for earlier work in identifying highly expressed and horizontally transferred genes in Escherichia coli. In this work, we applied Kohonen's self-organizing map to analysis of the codon usage pattern of the Escherichia coli, Aquifex aeolicus, Archaeoglobus fulgidus, Haemophilus influenzae RD:, Methanococcus jannaschii, Methanobacterium thermoautotrophicum, and Pyrococcus horikoshii genomes for evidence of highly expressed genes and horizontally transferred genes. All of the analyzed genomes had a clear category of horizontally transferred genes, and their apparent percentages ranged from 7.7% to 21.4%. The apparent percentage of highly expressed genes ranges from 0% to 11.8%. A clustering of average codon usage of main gene categories of the seven genomes showed an interesting mixing of gene classes in four thermophilic/hyperthermophilic organisms, A. aeolicus, A. fulgidus, M. thermoautotrophicum, and P. horikoshii, which suggests possible origins of their horizontally transferred genes as well as the need for adaptation to a specific environment. Further classification of the three gene categories in E. coli and H. influenzae according to gene function revealed that genes involved in communication (such as regulation and cell process) and structure (cell structure and structural proteins) are more likely to be horizontally transferred than are genes involved in information (transcription, translation, and related processes) and in some groups of energy (such as energy metabolism and carbon compound catabolism).

Archaeoglobus fulgidus↗
PubMed1999

New features and enhancements in the X-PLOR computer program.

This article describes new methods for X-ray crystallographic refinement and nuclear magnetic resonance (NMR) structure determination that are available in the recent release of the X-PLOR software, X-PLOR 98.0. The major new features of the X-PLOR 98.0 software are: (i) the introduction of maximum likelihood methods (Pannu and Read, Acta Crystallogr 1996;A52:659-668) for X-ray crystallographic refinement with structure factor amplitude, intensity and phase probability targets, (ii) the addition of the Andersen thermal coupling method for temperature control during simulated annealing refinements, (iii) a new utility function for converting reflection data in to the X-PLOR format, (iv) validated scripts and performance enhancements for structure determination from NMR distance restraints using torsion angle dynamics, (v) fast code for direct nuclear Oberhauser effect (NOE) refinement using matrix doubling and gaussian quadratures, (vi) methodologies for using ambiguous restraint information to perform automated iterative peak assignment and structure determination (Nilges et al., J Mol Biol 1997;269: 408-422). Additional developments in methodology for refining crystal structures from poor initial models include the implementation of a fast adaptive bulk solvent scattering correction and an energy minimization routine that makes use of second derivative information. Trial crystallographic refinements with an energy minimization protocol that includes these enhancements indicate significantly improved convergence. The quality of the resulting models appears comparable to models obtained from refinement protocols that incorporate torsion angle dynamics. Test applications of the new energy minimizer to NMR structure refinement with using NOE calculations also show improved convergence, leading to more optimized final models.

Crystallography, X-Ray↗
PubMed1998

Disproportionate fetal growth and fingerprint patterns.

Fingerprint whorl patterns are formed during fetal life. In a group of 180 term infants, those with more fingerprint whorls tended to have a small abdominal circumference (P = 0.09) and high ratio of head to abdominal circumference (P = 0.008). These associations were independent of the relation between the whorl counts of the mothers and their infants. We also found an independent correlation between the babies' whorl count and the combination of increasing subscapular (P = 0.03) and decreasing triceps (P = 0.02) skinfold thicknesses of the mothers. Whorl patterns are associated with adult hypertension; maternal nutritional status may influence their common origin during fetal development.

Adult↗
PubMed1996

Determination of protein and solvent volumes in protein crystals from contrast variation data.

By varying the relative values of protein and solvent scattering densities in a crystal, it is possible to obtain information on the shape and dimensions of protein molecular envelopes. Neutron diffraction methods are ideally suited to these contrast variation experiments because H/D exchange leads to large differential changes in the protein and solvent scattering densities and is structurally non-perturbing. Low resolution structure factors have been measured from cubic insulin crystals with differing H/D contents. Structure factors calculated from a simple binary density model, in which uniform scattering densities represent the protein and solvent volumes in the crystals, were compared with these data. The contrast variation differences in the sets of measured structure factors were found to be accurately fitted by this simple model. Trial applications to two problems in crystal structure determination illustrate how this fact may be exploited. (i) A translation function that employs contrast variation data gave a sharp minimum within 1-9A of the correctly positioned insulin molecule and is relatively insensitive to errors in the atomic model. (ii) An ab initio phasing method for the contrast variation data, based on analyzing histograms of the density distributions in trial maps, was found to recover the correct molecular envelope.

Citrate (si)-Synthase↗
PubMed1995

Neutron diffraction analysis of the solvent accessible volume in cubic insulin crystals.

The average contact distance between protein and solvent surface atoms in cubic insulin crystals has been determined from two sets of 15 A resolution neutron diffraction data. A contact distance between the water hydrogen sites and the protein surface that is significantly shorter than the average protein-water oxygen contact distance implies that many water molecules are oriented with hydrogen atoms pointed towards the protein surface. The shape of the protein/solvent interface is consistent with the protein envelope obtained from atomic co-ordinates.

Animals↗
PubMed1994

Thallium counterion distribution in cubic insulin crystals determined from anomalous x-ray diffraction data.

To determine the distribution of monovalent cations around a protein we have measured anomalous scattering diffraction data from Tl-containing cubic insulin crystals at pH 8 and pH 10.5. The differences between Bijvoet reflection pairs within each set of data were used to calculate anomalous scattering difference maps. Both maps show the same six Tl+ sites, which include two well-ordered Tl+ ions previously identified from isomorphous exchange experiments. The other four sites constitute a second class of cations, which, while much more mobile than the protein atoms, are associated with particular ligating groups. Three of the six Tl+ sites are created exclusively by protein main and side chain carbonyl dipoles rather than negatively charged groups. All of the Tl+ ions are positioned so as to interact with both protein atoms and water molecules. The Tl+ occupancies appear to depend in a complex way on interactions with each other and flexibility in the protein structure. The combined occupancies of these cations are slightly less than is required to neutralize the net protein charge of approximately -2e at pH 8 but account for only about half of the approximately -5e protein charge at pH 10.5. Thus, more disordered counterions, not seen in these Bijvoet anomalous scattering difference maps, are more numerous at higher protein net charge.

Animals↗
PubMed1994

Structure and selectivity of a monovalent cation binding site in cubic insulin crystals.

Cubic insulin crystals contain a binding site for monovalent cations in a cavity of the crystal dyad in which the bound cation is ligated by protein atomic dipoles and water molecules. These types of interaction are analogous to interactions that occur in small cation-selective carrier and channel molecules. X-ray diffraction data collected from cubic insulin crystals containing Li+, Na+, K+, NH4+, Rb+, and Tl+ show that (i) the differences in cation size do not cause any large alteration in the protein structure around the cation, and (ii) the bound cation is co-ordinated by one or two water molecules, depending on its ionic radii. The relative binding affinities for cations at this dyad site were obtained from an x-ray diffraction analysis of competition experiments in which crystals were dialyzed in mixtures of Tl+ with Li+, Na+, NH4+, Rb+, or Cs+. These data show that this site provides very little discrimination between Na+, K+, Rb+, and Tl+, some selectivity against the small Li+ and the tetrahedrally shaped NH4+, and stronger selectivity against the larger Cs+. The capacity of this site to bind monovalent cations of different sizes may be accounted for by the small number of protein ligating groups and a change from two ligating waters with Li+ and Na+ to one ligating water with the larger cations.

Animals↗
PubMed1993

Display and interpretation of solvent electron density distributions in insulin crystals.

In macromolecular crystallography, three-dimensional contour surfaces are useful for interactive computer graphics displays of the protein electron density but are less effective for presenting static images of large volumes of solvent density. A raster-based computer graphics program which displays depth-cued projections of continuous density distributions has been developed to analyze the distribution of solvent atoms in macromolecular crystals. Maps of the water distribution in the cubic insulin crystal show some well-ordered waters, which are bound to surrounding protein atoms by multiple hydrogen bonds, and an ill-defined solvent structure at a greater distance from the protein surface. Molecular dynamics calculations were used to assist in the interpretation of the time-varying solvent structure within two enclosed cavities in the crystal. Two water molecules that ligate a sodium ion were almost immobile during the stimulation but the majority of water molecules were found to move rapidly between the density maxima identified from the crystallographic refinement.

Animals↗
PubMed1993

Multiple hydration layers in cubic insulin crystals.

Cubic insulin crystals contain approximately 30-A-diameter channels filled with aqueous solvent, providing a useful system in which to analyze hydration structure at a variety of distances from protein surfaces. Beginning with an atomic model for the protein and ordered water molecules, the density distribution in the solvent volume of the phasing model was iteratively refined to improve the fit of calculated structure factors with x-ray diffraction data. The free R value, which compares calculated structure factors with a subset of observed structure factors deliberately omitted from the refinement, was used to provide an objective confirmation of the effectiveness of the refinement procedure. Electron density maps of the solvent, computed using the solvent-refined phases and complete low-resolution diffraction data, reveal multiple hydration layers around the protein.

Animals↗
PubMed1992

Conformational changes in cubic insulin crystals in the pH range 7-11.

To determine the effect of variations in the charge distribution on the conformation of a protein molecule, we have solved the structures of bovine cubic insulin over a pH range from 7 to 11 in 0.1 M and 1 M sodium salt solutions. The x-ray data were collected beyond 2-A resolution and the R factors for the refined models ranged from 0.16 to 0.20. Whereas the positions of most protein and well-ordered solvent atoms are conserved, about 30% of residues alter their predominant conformation as the pH is changed. Conformational switching of A5 Gln and B10 His correlates with the pH dependence of monovalent cation binding to insulin in cubic crystals. Shifts in the relative positions of the A chain NH2-terminal and B chain COOH-terminal groups are probably due to titration of the A1 alpha-amino group. Two alternative positions of B25 Phe and A21 Asn observed in cubic insulin at pH 11 are similar to those found in two independent molecules of the 2Zn insulin dimer at pH 6.4. The conformational changes of the insulin amino acids appear to be only loosely coupled at distant protein sites. Shifts in the equilibrium between distinct conformational substates as the charge distribution on the protein is altered are analogous to the electrostatically triggered movements that occur in many functional protein reactions.

Animals↗
PubMed1992

Monovalent cation binding to cubic insulin crystals.

Two localized monovalent cation binding sites have been identified in cubic insulin from 2.8 A-resolution difference electron density maps comparing crystals in which the Na+ ions have been replaced by Tl+. One cation is buried in a closed cavity between insulin dimers and is stabilized by interaction with protein carbonyl dipoles in two juxtaposed alternate positions related by the crystal dyad. The second cation binding site, which also involves ligation with carbonyl dipoles, is competitively occupied by one position of two alternate His B10 side chain conformations. The cation occupancy in both sites depends on the net charge on the protein which was varied by equilibrating crystals in the pH range 7-10. Detailed structures of the cation binding sites were inferred from the refined 2-A resolution map of the sodium-insulin crystal at pH 9. At pH 9, the localized monovalent cations account for less than one of the three to four positive counterion charges necessary to neutralize the negative charge on each protein molecule. The majority of the monovalent counterions are too mobile to show up in the electron density maps calculated using data only at resolution higher than 10 A. Monovalent cations of ionic radius less than 1.5 A are required for crystal stability. Replacing Na+ with Cs+, Mg++, Ca++ or La+++ disrupts the lattice order, but crystals at pH 9 with 0.1 M Li+, K+, NH4+, Rb+ or Tl+ diffract to at least 2.8 A resolution.

Animals↗
PubMed1992

Flexibility in crystalline insulins.

Comparisons of atomic models for chemically identical protein molecules solved in differing crystal environments provide information on flexibility in the protein structure. The structures of five T4 lysozyme proteins in differing crystal environments showed large relative displacements of the two domains with conserved backbone conformations that are connected by a flexible hinge (H. R. Faber and B. W. Matthews. 1990. Nature (Lond.). 348:263-266). In contrast, my comparison of the positions of all the atoms in two crystal forms of insulin shows that the structural changes caused by the differing crystal contacts are contained within nearby amino acids and are not propagated through the core of the insulin molecule. Groups of atoms that are most significantly displaced are not shifted in large rigid units but are repacked into new and distinct conformations. The transmission of displacements through the single domain insulin molecule is, like the movements due to thermal vibrations (D. L. D. Caspar, J. Clarage, D. M. Salunke, M. S. Clarage. 1988. Nature (Lond.). 332:659-662), characterized by short-range interactions between small atomic groups.

Insulin↗
PubMed1992

Unilateral subungual hyperkeratosis following a cerebrovascular incident in a patient with psoriasis.

The prevalence of nail abnormalities in psoriasis is reported to be between 50 and 90% of affected adults. These include pitting, nail discoloration, onycholysis and subungual hyperkeratosis. Although nail changes may be associated with neurological disease, there are no previous reports of psoriatic nail changes following cerebrovascular events. We report the first case to our knowledge of unilateral subungual hyperkeratosis in a psoriatic patient which developed following a cerebrovascular event.

Cerebrovascular Disorders↗
PubMed1991

Structure of the pig insulin dimer in the cubic crystal.

Atomic coordinates for pig insulin in the cubic crystal have been refined by reciprocal-space methods to an R factor of 0.173 for data between 10.0 and 1.7 A resolution with structure-factor amplitudes greater than two standard deviations. Stereochemical parameters for the refined model are close to standard values and the estimated error in the positions of well-ordered atoms is about 0.1 A. Residues directly involved in the formation of the exact (crystallographic) cubic insulin dimer are oriented similarly to those in the non-crystallographic 2Zn insulin dimer. Other residues, which make different molecular contacts in the different crystal forms, have locally altered conformations. The cubic insulin molecule is significantly more similar to one of the two independent molecules in the 2Zn insulin dimer than the other. This more similar molecule is expected to be the more stable conformer.

Animals↗