Real space refinement of neutron diffraction data from sperm whale carbonmonoxymyoglobin

Neutron diffraction data from crystals of sperm whale carbonmonoxymyoglobin have been refined by the real space refinement technique. Estimates of the neutron occupancies at the end of the refinement show that the mean for each atom type (including hydrogen and deuterium) is close to the expected value and has a standard deviation from the mean of about 5%. Mean neutron occupancies of main-chain atoms involved in deuterium bonds versus those not involved in deuterium bonds demonstrate that the hydrogen/deuterium exchange of the latter group is higher. The oxygen and deuterium co-ordinates for 40 water molecules have been determined: 27 of these water molecules were involved in bridges between protein atoms, and nine were involved in deuterium bonds with main-chain atoms. The deuterium-bond angles in helical regions show significant deviations from linearity. The mean ND … O angle was 154(3) °² and the mean CO … D angle was 145(3) °.     

Automated interpretation of electron density maps as applied to Bence-Jones protein Rhe

Previous results obtained from the computerized interpretation (Greer, 1976) of a 3.0 Å electron density map of Bence—Jones protein Rhe have been compared with results (Wang et al., 1979) obtained by classical Richards' box techniques (Richards, 1968). Although the overall agreement between the two models is generally good, the computerized results contain several significant errors in the assignment of α-carbon atoms, which in our opinion are unlikely to be corrected without using human intervention together with other methods. However this test does show that the computerized method has potential.     

A map of the sites on bacteriophage PM2 DNA for the restriction endonucleases HindIII and HpaII

The map of the seven sites for the restriction endonuclease HindIII³ and the single site for endo R.HpaII on PM2 DNA was determined. This map was oriented with respect to the denaturation map of this DNA (Brack et al., 1975) by partial denaturation mapping of the fragments. A new method for localizing restriction fragments by DNA-DNA hybridization and electron microscopy is described.     

Crystallographic study of turkey egg-white lysozyme and its complex with a disaccharide

The crystal structure of turkey egg-white lysozyme, determined by the molecular replacement method at 5 Å resolution (Bott & Sarma, 1976) has now been refined to 2.8 Å resolution and a model has been built to fit the electron density. A comparison of the co-ordinates with those of hen lysozyme indicate a rootmean-square deviation of 1.6 Å for all the main-chain and side-chain atoms. A significant difference is observed in the region of residues 98 to 115 of the structure. The molecules are packed in this crystal form with the entire length of the active cleft positioned in the vicinity of the crystallographic 6-fold axis and is not blocked by neighboring molecules. A difference electron density map calculated between crystals of turkey lysozyme soaked in a disaccharide of N-acetyl glucosamine—N-acetyl muramic acid and the native crystals showed a strong positive peak at subsite C, a weak positive peak at subsite D and two strong peaks that correspond to the subsite E and a new subsite F′. This new site F′ is different from the subsite F predicted for the sixth saccharide from model building in hen lysozyme. The interactions between the saccharides bound at subsites E and F′ and the enzyme molecules are discussed.     

Sequencing a protein by x-ray crystallography. II. Refinement of yeast hexokinase B co-ordinates and sequence at 2.1 A resolution.

Although the amino acid sequence of yeast hexokinase B has not been determined by chemical means, crystallographic refinement of the hexokinase monomer was carried out at 2.1 Å resolution to improve both the atomic co-ordinates and the amino acid sequence, which had been obtained from a 2.5 Å electron density map. The atomic co-ordinates were adjusted by real-space refinement into a multiple isomorphous replacement map, followed by automated difference Fourier refinement, and restrained parameter structure factor least-squares refinement. The amino acid sequence was altered periodically after visual inspection of (F_o ? F_c) difference electron density maps. Evidence of the improvement in the amino acid sequence was provided by the better agreement between the X-ray and chemically derived amino acid compositions, and most importantly by the ability to locate two short peptides which had been chemically sequenced. While only 6 out of the 18 residues in these two peptides agree with the sequence of the original model, 12 residues agree with the sequence of the refined model and the others differ by only an atom or two. The refined model contains 3293 of of the 3596 non-hydrogen atoms expected from the amino acid composition and 152 bound water molecules. The crystallographic R factor at 2.1 Å is 0.25.We show that there are several advantages to refining the structure of even a protein of unknown sequence. (1) Improved phases can be obtained to the resolution limit of the diffraction pattern starting with a model derived from a 2.5 Å map. (2) The accuracy of the amino acid sequence derived by X-ray methods alone can be substantially improved. (3) Functionally important residues can be identified before chemical sequence information is available. (4) The improved X-ray sequence should greatly reduce the effort required to obtain a chemical sequence; since peptides as short as eight or nine residues can be located in the refined X-ray sequence, peptides do not need to be overlapped by chemical means.     

A low-resolution study of testicular lactate dehydrogenase using the molecular replacement technique

The orientation of the molecular 2-fold axes of mouse testicular lactate dehydrogenase (LDHase³-C₄) was determined by a rotation function search. These were subsequently identified with the P, Q, and R axes of dogfish LDHase-M₄. Since LDHase-C₄ crystallized with one molecule in a triclinic cell, the origin of the co-ordinate system was arbitrarily fixed at the molecular center. Structure factor phases were derived from an appropriately oriented dogfish apo LDHase-M₄ phasing model and combined with the observed structure amplitudes to produce a hybrid electron density map. Density points related by the molecular 222 point symmetry were averaged so as to remove the bias of the phasing model. At 7.5 Å resolution, the structure of the crystallized mouse LDHase-C₄ was found to be without coenzyme, with a conformation indistinguishable from that of dogfish apo LDHase-M₄.     

Plasma diagnostics from intensities of resonance line series of He-like ions

The possibility of using the relative intensities of the 1snp ¹P₁–1s ^{2 1}S0 transitions with n = 3–6 in He-like multicharged ions to diagnose plasma in a nonstationary ionization state is considered. The calculations performed for F VIII ions show that, at electron temperatures of T _e = 10–100 eV, the intensity ratios are sensitive to the plasma electron density in the range of N _e = 10¹⁶–10²⁰ cm^–3. The universal calculated dependences can be used to diagnose various kinds of recombining or ionizing plasmas containing such ions.     

Comparison of automated and conventional microbiological examination of donated human cardiac tissue in heart valve banking

Most tissue banks use the conventional method; however, the automated method has advantages over the conventional method. The aim of this study was to compare the conventional and automated methods of culture in human cardiac tissue using an artificial contamination model. Myocardial samples were contaminated with sequential concentration (10⁴ to 10^?1 CFU/mL) with Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Candida albicans. Cultures were obtained from solution were the fragment was immersed and minced tissue, before and after the routine decellularization solution, with automated and conventional culture methods. Automated and conventional methods were compared and a p value ≤?0.05 was considered significant. Staphylococcus aureus presented a significantly higher growth in the automated method, as well as faster than the conventional (p?<?0.05). The positivity for growth in the automated method was higher in concentrated inoculum (>?10² CFU/mL) (p?<?0.05). The growth in the automated method was significantly faster than conventional when inoculum concentration was above 10³ CFU/mL. The automated culture method is faster than conventional method with a higher positivity in a contaminated model of myocardial and transport solution used in tissue banks.     

Electronic fine structure calculation of metal complexes with three-open-shell <Emphasis Type="Italic">s</Emphasis>, <Emphasis Type="Italic">d,and p</Emphasis> configurations

The ligand field density functional theory (LFDFT) algorithm is extended to treat the electronic structure and properties of systems with three-open-shell electron configurations, exemplified in this work by the calculation of the core and semi-core 1s, 2s, and 3s one-electron excitations in compounds containing transition metal ions. The work presents a model to non-empirically resolve the multiplet energy levels arising from the three-open-shell systems of non-equivalent ns, 3d, and 4p electrons and to calculate the oscillator strengths corresponding to the electric-dipole 3d ^m  → ns ¹3d ^m 4p ¹ transitions, with n = 1, 2, 3 and m = 0, 1, 2, …, 10 involved in the s electron excitation process. Using the concept of ligand field, the Slater-Condon integrals, the spin-orbit coupling constants, and the parameters of the ligand field potential are determined from density functional theory (DFT). Therefore, a theoretical procedure using LFDFT is established illustrating the spectroscopic details at the atomic scale that can be valuable in the analysis and characterization of the electronic spectra obtained from X-ray absorption fine structure or electron energy loss spectroscopies.     

Crystal structure of Bence Jones protein rhe (3 A) and its unique domain-domain association.

The crystal structure of protein Rhe, a lambda type V_L dimer, has been determined at a resolution of 3 Å by the method of multiple isomorphous replacement supplemented with anomalous scattering data. A crystallographic sequence was assigned from an interpretation of the electron density map in an optical comparator and is compared with a chemically determined partial amino acid sequence. The monomeric unit of Rhe, as determined crystallographically, contains 113 amino acids, 110 belonging to the variable region and three belonging to the constant segment of a light chain. The single polypeptide chain constituting the monomer forms a nine-stranded β-barrel characteristic of V domains. The β-pleated sheet surrounds an ellipsoidally shaped interior core of approximately 10 Å × 15 Å × 25 Å in size. The monomers that are related by the crystallographic dyad are held together as dimers by interdomain hydrogen bonds and hydrophobic interactions. At one end of the dimer is an opening which is lined exclusively by residues from the hypervariable regions.A comparison of Rhe with Rei, a kappa type V_L dimer (Epp et al., 1975), revealed that monomers of Rhe and Rei dimerized differently. Their respective dyad and pseudodyad of dimerization are not the same, and this causes a variation in the overall steric arrangement of the hypervariable regions in the two cavities. In adition a dissimilarity was observed in the non-hypervariable segment linking the first and second hypervariable regions. This segment is in the form of a loop and it includes most of the residues participating in the interdomain interactions stabilizing dimer formation in both proteins and these loop positions differ by as much as 7 Å. Our results also show that there is a good correlation between the dissimilarity of the loop position and the difference in the domain-association. Our preliminary analysis indicates that the positions of the corresponding non-hypervariable loops in V domains may be determined in part by the residues in the hypervariable regions.Accordingly, the three-dimensional structure of Rhe suggests that this nonhypervariable loop in V_L and its counterpart in V_H may have an important biological function in antibody specificity and variability by virtue of their influence over the architecture of the complementarity site.     

Construction of an ultra-high density consensus genetic map,and enhancement of the physical map from genome sequencing in <Emphasis Type="Italic">Lupinus angustifolius</Emphasis>

Key message

An ultra-high density genetic map containing 34,574 sequence-defined markers was developed in Lupinus angustifolius. Markers closely linked to nine genes of agronomic traits were identified. A physical map was improved to cover 560.5 Mb genome sequence.

Abstract

Lupin (Lupinus angustifolius L.) is a recently domesticated legume grain crop. In this study, we applied the restriction-site associated DNA sequencing (RADseq) method to genotype an F₉ recombinant inbred line population derived from a wild type × domesticated cultivar (W × D) cross. A high density linkage map was developed based on the W × D population. By integrating sequence-defined DNA markers reported in previous mapping studies, we established an ultra-high density consensus genetic map, which contains 34,574 markers consisting of 3508 loci covering 2399 cM on 20 linkage groups. The largest gap in the entire consensus map was 4.73 cM. The high density W × D map and the consensus map were used to develop an improved physical map, which covered 560.5 Mb of genome sequence data. The ultra-high density consensus linkage map, the improved physical map and the markers linked to genes of breeding interest reported in this study provide a common tool for genome sequence assembly, structural genomics, comparative genomics, functional genomics, QTL mapping, and molecular plant breeding in lupin.

     

X-ray crystallographic studies of seal myoglobin: The molecule at 2.5 Å resolution

The three-dimensional structure of metmyoglobin from the common seal has been determined at 2.5 Å resolution. The isomorphous replacement technique has been employed using two derivatives, the mercuri-iodide and the aurichloride. Four-circle diffractometer data to a Bragg angle θ = 18.05 ° were measured for one complete set of Friedel pairs of reflexions from each type of protein crystal. Atomic positions for the individual atoms in the (HgI^?₃₎-group at the two sites of attachment were obtained from three-dimensional difference electron density maps and were further refined. A ‘best’ electron density map of the native protein based on refined heavy-atom parameters was interpreted with the help of the known amino acid sequence, and co-ordinates for all the non-hydrogen atoms were measured from the model. Those of the globin were further constrained according to the ‘modelfit’ procedure of Dodson et al. (1976). The molecule is described in detail; the conformations of the side-chains relative to the positions of the heavy atoms and to the interface between neighbouring molecules are discussed. A preliminary residue by residue comparison of the seal and sperm whale myoglobin molecules is presented in the accompanying paper.     

Oxidation of glucose by syntrophic association between <Emphasis Type="Italic">Geobacter</Emphasis> and hydrogenotrophic methanogens in microbial fuel cell

Objective

To investigate a syntrophic interaction between Geobacter sulfurreducens and hydrogenotrophic methanogens in sludge-inoculated microbial fuel cell (MFC) systems running on glucose with an improved electron recovery at the anode.

Results

The presence of archaea in MFC reduces Coulombic efficiency (CE) due to their electron scavenging capability but, here, we demonstrate that a syntrophic interaction can occur between G. sulfurreducens and hydrogenotrophic methanogens via interspecies H₂ transfer with improvement in CE and power density. The addition of the methanogenesis inhibitor, 2-bromoethanesulfonate (BES), resulted in the reduction in power density from 5.29 to 2 W/m³, and then gradually increased to the peak value of 5.5 W/m³ when BES addition was stopped.

Conclusion

Reduction of H₂ partial pressure by archaea is an efficient approach in improving power output in a glucose-fed MFC system using Geobacter sp. as an inoculum.

     

Axial arrangement of crossbridges in thick filaments of vertebrate skeletal muscle.

X-ray intensity data to 1.8 Å resolution were collected from native trigonal crystals of bovine trypsinogen. The orientation and position of the trypsinogen molecules within their crystal cells were determined by Patterson search techniques using the refined model of bovine trypsin (Bode &; Schwager, 1975), and by subsequent R factor refinement. The translation functions allowed discrimination between the enantiomorphic space groups P3₂21 and P3₁21. After one constrained crystallographic refinement cycle, which reduced the crystallographic reliability factor (R) from 35% to 31%, a preliminary difference Fourier map showed several interesting details. Several refinement cycles reduced the value of R to 23%. The overall chain folding is very similar to trypsin. The chain segments, including residues 184 to 193² and 217 to 223, which form the specificity pocket in trypsin, are flexible in trypsinogen. The autolysis loop is partially mobile between residues 142 and 152. There is no continuing electron density for the N terminal residues preceding Tyr20. This indicates that the N terminus may be only weakly fixed to the rest of the molecule or may even float freely in solution.     

Interferometric studies of the electron density dynamics at the periphery of a micropinch discharge

Results are presented from experimental studies of the electron density dynamics at the periphery of a low-inductance vacuum spark. The measurements were performed using a newly developed homodyne quadrature interferometer possessing fairly high dynamic range and performance. The experiments demonstrated the presence of a plasma with an electron density of n _e ≥ 10¹⁸ cm^?3 at the periphery of the discharge gap. The radial electron density distribution was shown to have a pronounced tubular structure. The relatively high values of the peripheral electron density indicate that the process of pinching can be substantially affected by shunt currents flowing at the discharge periphery.     

Area and Volumetric Density Estimation in Processed Full-Field Digital Mammograms for Risk Assessment of Breast Cancer

Introduction

Mammographic density, the white radiolucent part of a mammogram, is a marker of breast cancer risk and mammographic sensitivity. There are several means of measuring mammographic density, among which are area-based and volumetric-based approaches. Current volumetric methods use only unprocessed, raw mammograms, which is a problematic restriction since such raw mammograms are normally not stored. We describe fully automated methods for measuring both area and volumetric mammographic density from processed images.

Methods

The data set used in this study comprises raw and processed images of the same view from 1462 women. We developed two algorithms for processed images, an automated area-based approach (CASAM-Area) and a volumetric-based approach (CASAM-Vol). The latter method was based on training a random forest prediction model with image statistical features as predictors, against a volumetric measure, Volpara, for corresponding raw images. We contrast the three methods, CASAM-Area, CASAM-Vol and Volpara directly and in terms of association with breast cancer risk and a known genetic variant for mammographic density and breast cancer, rs10995190 in the gene ZNF365. Associations with breast cancer risk were evaluated using images from 47 breast cancer cases and 1011 control subjects. The genetic association analysis was based on 1011 control subjects.

Results

All three measures of mammographic density were associated with breast cancer risk and rs10995190 (p<0.025 for breast cancer risk and p<1×10⁻⁶ for rs10995190). After adjusting for one of the measures there remained little or no evidence of residual association with the remaining density measures (p>0.10 for risk, p>0.03 for rs10995190).

Conclusions

Our results show that it is possible to obtain reliable automated measures of volumetric and area mammographic density from processed digital images. Area and volumetric measures of density on processed digital images performed similar in terms of risk and genetic association.     

A near atomic resolution structure of a Melanocarpus albomyces laccase

It is becoming routine for cryoEM single particle reconstructions to result in 3D electron density maps with resolutions of 10 Å, but maps with resolutions of 5 Å or better are still celebrated events. The electron microscope has a resolving power to better than 2 Å, and thus should not be a limiting factor; instead the practical limitations in resolution most likely arise from a combination of specimen preparation methods, data collection parameters, and data analysis procedures. With the aid of a highly automated system for acquiring images, coupled to a relational database to keep track of all processing parameters, we have taken a systematic approach to optimizing parameters affecting the resolution of single particle reconstructions. Using GroEL as a test-bed, we performed a series of 3D reconstructions where we systematically varied the number of particles used in computing the map, the accelerating voltage of the microscope, and the electron dose used to acquire the images. We also investigated methods for excluding unacceptable or “bad” particles from contributing to the final 3D map. Using relatively standard instrumentation (Tecnai F20, 4K × 4K CCD, side entry cold stage) and a completely automated approach, these approaches resulted in a map with a nominal resolution of 5.4 Å (FSC_0.5) in which secondary structure is clearly discernable and the handedness of some of the α-helices in the GroEL structure can be determined.     

Fully Automated Pulmonary Lobar Segmentation: Influence of Different Prototype Software Programs onto Quantitative Evaluation of Chronic Obstructive Lung Disease

Objectives

Surgical or bronchoscopic lung volume reduction (BLVR) techniques can be beneficial for heterogeneous emphysema. Post-processing software tools for lobar emphysema quantification are useful for patient and target lobe selection, treatment planning and post-interventional follow-up. We aimed to evaluate the inter-software variability of emphysema quantification using fully automated lobar segmentation prototypes.

Material and Methods

66 patients with moderate to severe COPD who underwent CT for planning of BLVR were included. Emphysema quantification was performed using 2 modified versions of in-house software (without and with prototype advanced lung vessel segmentation; programs 1 [YACTA v.2.3.0.2] and 2 [YACTA v.2.4.3.1]), as well as 1 commercial program 3 [Pulmo3D VA30A_HF2] and 1 pre-commercial prototype 4 [CT COPD ISP ver7.0]). The following parameters were computed for each segmented anatomical lung lobe and the whole lung: lobar volume (LV), mean lobar density (MLD), 15^th percentile of lobar density (15^th), emphysema volume (EV) and emphysema index (EI). Bland-Altman analysis (limits of agreement, LoA) and linear random effects models were used for comparison between the software.

Results

Segmentation using programs 1, 3 and 4 was unsuccessful in 1 (1%), 7 (10%) and 5 (7%) patients, respectively. Program 2 could analyze all datasets. The 53 patients with successful segmentation by all 4 programs were included for further analysis. For LV, program 1 and 4 showed the largest mean difference of 72 ml and the widest LoA of [-356, 499 ml] (p<0.05). Program 3 and 4 showed the largest mean difference of 4% and the widest LoA of [-7, 14%] for EI (p<0.001).

Conclusions

Only a single software program was able to successfully analyze all scheduled data-sets. Although mean bias of LV and EV were relatively low in lobar quantification, ranges of disagreement were substantial in both of them. For longitudinal emphysema monitoring, not only scanning protocol but also quantification software needs to be kept constant.