A systematic comparison of three structure determination methods from NMR data: Dependence upon quality and quantity of data

Summary We have systematically examined how the quality of NMR protein structures depends on (1) the number of NOE distance constraints. (2) their assumed precision, (3) the method of structure calculation and (4) the size of the protein. The test sets of distance constraints have been derived from the crystal structures of crambin (5 kDa) and staphylococcal nuclease (17 kDa). Three methods of structure calculation have been compared: Distance Geometry (DGEOM), Restrained Molecular Dynamics (XPLOR) and the Double Iterated Kalman Filter (DIKF). All three methods can reproduce the general features of the starting structure under all conditions tested. In many instances the apparent precision of the calculated structure (as measured by the RMS dispersion from the average) is greater than its accuracy (as measured by the RMS deviation of the average structure from the starting crystal structure). The global RMS deviations from the reference structures decrease exponentially as the number of constraints is increased, and after using about 30% of all potential constraints, the crrors asymptotically approach a limiting value. Increasing the assumed precision of the constraints has the same qualitative effect as increasing the number of constraints. For comparable numbers of constraints/residue, the precision of the calculated structure is less for the larger than for the smaller protein, regardless of the method of calculation. The accuracy of the average structure calculated by Restrained Molecular Dynamics is greater than that of structures obtained by purely geometric methods (DGEOM and DIKF).     

Statistical torsion angle potential energy functions for protein structure modeling: A bicubic interpolation approach

A set of grid type knowledge‐based energy functions is introduced for ?–χ₁, ψ–χ₁, ?–ψ, and χ₁–χ₂ torsion angle combinations. Boltzmann distribution is assumed for the torsion angle populations from protein X‐ray structures, and the functions are named as statistical torsion angle potential energy functions. The grid points around periodic boundaries are duplicated to force periodicity, and the remedy relieves the derivative discontinuity problem. The devised functions rapidly improve the quality of model structures. The potential bias in the functions and the usefulness of additional secondary structure information are also investigated. The proposed guiding functions are expected to facilitate protein structure modeling, such as protein structure prediction, protein design, and structure refinement. Proteins 2013. Proteins 2013; 81:1156–1165. © 2013 Wiley Periodicals, Inc.     

On the NMR structure determination of a 44n RNA pseudoknot: Assignment strategies and derivation of torsion angle restraints

The complete T- and pseudoknotted acceptor arm of the tRNA-like structure of turnip yellow mosaic virus (TYMV) genomic RNA has been studied by NMR spectroscopy. Resonance assignment and the gathering of restraints of the 44-mer are impeded by spectral complexity as well as by line broadening. The latter is caused by local dynamical effects in the pseudoknot domain in the molecule. These specific problems could be solved by using different field strengths and selectively ¹³C/¹⁵ labeled samples. Experiments for assigning the sugar spin systems were adjusted to satisfy the requirements of this system. Furthermore, the quality of the structure could be improved by determining the backbone torsion angles , and , using new approaches that were tailored for use in large RNA molecules.     

Relaxation data in NMR structure determination: model calculations for the lysozyme-Gd3+ complex

The effect of including paramagnetic relaxation data as additional restraints in the determination of protein tertiary structures from NMR data has been explored by a systematic series of model calculations. The system used for testing the method was the 2.0 A resolution tetragonal crystal structure of hen egg white lysozyme (129 amino acid residues) and structures were generated using a version of the hybrid "distance geometry-dynamic simulated annealing" procedure. A limited set of 769 NOEs was used as restraints in all the calculations; the strengths of these were categorized into three classes on the basis of distances observed in the crystal structure. The values of 50 phi angles were also restrained on the basis of amide-alpha coupling constants calculated from the X-ray structure. Five sets of 12 structures were determined using differing sets of paramagnetic relaxation data as restraints additional to those involving the NOE and coupling constant data. The paramagnetic relaxation data were modeled on the basis of the distances of defined protons from the crystallographic binding site of Gd3+ in lysozyme. Analysis of the results showed that the relaxation data significantly improved the correspondence between the set of generated structures and the crystal structure, and that the more well defined the relaxation data, the more significant the improvement in the quality of the structures. The results suggest that the inclusion of paramagnetic relaxation restraints could be of significant value for the experimental determination of protein structures from NMR data.     

MUNIN: A new approach to multi-dimensional NMR spectra interpretation

A new method, MUNIN (Multi-dimensional NMR spectra interpretation), is introduced for the automated interpretation of three-dimensional NMR spectra. It is based on a mathematical concept referred to as three-way decomposition. An NMR spectrum is decomposed into a sum of components, with each component corresponding to one or a group of peaks. Each component is defined as the direct product of three one-dimensional shapes. A consequence is reduction in dimensionality of the spectral data used in further analysis. The decomposition may be applied to frequency-domain or time-domain data, or to a mixture of these. Features of MUNIN include good resolution in crowded regions and the absence of assumptions about line shapes. Uniform sampling of time-domain data, a prerequisite for discrete Fourier transform, is not required. This opens an avenue for the processing of NMR data that do not follow oscillating behaviour, e.g. from relaxation measurements. The application of MUNIN is illustrated for a ¹H-¹⁵N-NOESY-HSQC, where each component is defined as the set of all NOE peaks formed by a given amide group. As a result, the extraction of structural information simply consists of one-dimensional peak picking of the shape along the NOE-axis obtained for each amide group.     

Indirect determination of magnetic susceptibility tensors in peroxidases: a novel approach to structure elucidation by NMR

 The chemical shifts of several ¹³C nuclei positioned α to the haems in oxidised cyanide complexes of horseradish peroxidase and lignin peroxidase are reported and analysed in terms of π molecular orbitals with perturbed D_4h symmetry. The additional contributions to the paramagnetic shifts of ¹³C nuclei in the vinyl groups which arise from conjugation with the porphyrin π molecular orbitals are discussed, and an empirical correction factor is derived from a number of other compounds which contain haems b. The orbital mixing parameter which is obtained from the analysis of the experimental ¹³C shifts is compared with the orientation of the axial histidine ligands in X-ray structures of related compounds and found to be close to the orientation of the normal to the histidine ring. Comparison with the magnetic axes determined by fitting the dipolar shifts of several protons which have been assigned previously also shows close agreement with the negative in-plane rotation of the magnetic y axis. It is therefore possible to obtain the approximate orientation of the magnetic axes from ¹³C resonances of the haem and hence to determine the dipolar shifts at any point in space with respect to the haem by using these axes together with the anisotropy of the magnetic susceptibility, which can be obtained by extrapolation from EPR g values. Excellent agreement is found between dipolar shifts obtained by fitting an empirical magnetic susceptibility tensor and predictions based on ¹³C NMR and EPR in the case of lignin peroxidase. The agreement is less good in the case of horseradish peroxidase, in which the empirical magnetic z axis appears to be tilted significantly away from the haem normal, though this may be due in part to the lack of accurate atomic coordinates. It is concluded that useful estimates of the magnetic susceptibility tensor may be obtained from ¹³C NMR and EPR studies even in large mammalian peroxidases for which no structural models are available. Received: 27 December 1995 / Accepted: 17 April 1996     

Towards structure determination of neurotoxin II bound to nicotinic acetylcholine receptor: a solid-state NMR approach

Solid-state magic-angle spinning nuclear magnetic resonance (NMR) has sufficient resolving power for full assignment of resonances and structure determination of immobilised biological samples as was recently shown for a small microcrystalline protein. In this work, we show that highly resolved spectra may be obtained from a system composed of a receptor-toxin complex. The NMR sample used for our studies consists of a membrane preparation of the nicotinic acetylcholine receptor from the electric organ of Torpedo californica which was incubated with uniformly 13C-,15N-labelled neurotoxin II. Despite the large size of the ligand-receptor complex ( > 290 kDa) and the high lipid content of the sample, we were able to detect and identify residues from the ligand. The comparison with solution NMR data of the free toxin indicates that its overall structure is very similar when bound to the receptor, but significant changes were observed for one isoleucine.     

A high quality nuclear magnetic resonance solution structure of peptide deformylase from Escherichia coli: Application of an automated assignment strategy using GARANT

The NMR structure of the peptide deformylase (PDF) (1–150) from Escherichia coli, which is an essential enzyme that removes the formyl group from nascent polypeptides and represents a potential target for drug discovery, was determined using 15N/13C doubly labeled protein. Nearly completely automated assignment routines were employed to assign three-dimensional triple resonance, 15N-resolved and 13C-resolved NOESY spectra using the program GARANT. This assignment strategy, demonstrated on a 17 kDa protein, is a significant advance in the automation of NMR data assignment and structure determination that will accelerate future work. A total of 2302 conformational constraints were collected as input for the distance geometry program DYANA. After restrained energy minimization with the program X-PLOR the 20 best conformers characterize a high quality structure with an average of 0.43 Å for the root-mean-square deviation calculated from the backbone atoms N, C and C, and 0.81 Å for all heavy atoms of the individual conformers relative to the mean coordinates for residues 1 to 150. The globular fold of PDF contains two -helices comprising residues 25–40, 125–138, six -strands 57–60, 70–77, 85–88, 98–101, 105–111, 117–123 and one 310 helix comprising residues 49–51. The C-terminal helix contains the HEXXH motif positioning a zinc ligand in a similar fashion to other metalloproteases, with the third ligand being cysteine and the fourth presumably a water. The three-dimensional structure of PDF affords insight into the substrate recognition and specificity for N-formylated over N-acetylated substrates and is compared to other PDF structures.     

From principles to practice: a spatial approach to systematic conservation planning in the deep sea

Increases in the demand and price for industrial metals, combined with advances in technological capabilities have now made deep-sea mining more feasible and economically viable. In order to balance economic interests with the conservation of abyssal plain ecosystems, it is becoming increasingly important to develop a systematic approach to spatial management and zoning of the deep sea. Here, we describe an expert-driven systematic conservation planning process applied to inform science-based recommendations to the International Seabed Authority for a system of deep-sea marine protected areas (MPAs) to safeguard biodiversity and ecosystem function in an abyssal Pacific region targeted for nodule mining (e.g. the Clarion–Clipperton fracture zone, CCZ). Our use of geospatial analysis and expert opinion in forming the recommendations allowed us to stratify the proposed network by biophysical gradients, maximize the number of biologically unique seamounts within each subregion, and minimize socioeconomic impacts. The resulting proposal for an MPA network (nine replicate 400 × 400 km MPAs) covers 24% (1 440 000 km²) of the total CCZ planning region and serves as example of swift and pre-emptive conservation planning across an unprecedented area in the deep sea. As pressure from resource extraction increases in the future, the scientific guiding principles outlined in this research can serve as a basis for collaborative international approaches to ocean management.     

Multi-conformational peptide dynamics derived from NMR data: A new search algorithm and its application to antamanide

Summary A search algorithm, called MEDUSA, is presented which allows the determination of multiple conformations of biomolecules in solution with exchange rate constants typically between 10³ and 10⁷ s^–1 on the basis of experimental high-resolution NMR data. Multiples of structures are generated which are consistent as ensembles with NMR cross-relaxation rates (NOESY, ROESY), scalar J-coupling constants, and T_1p measurements. The algorithm is applied to the cyclic decapeptide antamanide dissolved in chloroform. The characteristic radio-frequency field dependence of the T_1p relaxation rates found for the NH protons of Val¹ and Phe⁶ can be explained by a dynamical exchange between two structures.     

A high-resolution 1H NMR approach for structure determination of membrane peptides and proteins in non-deuterated detergent: Application to mastoparan X solubilized in n-octylglucoside

Summary Application of ¹H 2D NMR methods to solubilized membrane proteins and peptides has up to now required the use of selectively deuterated detergents. The unavailability of any of the common biochemical detergents in deuterated form has therefore limited to some extent the scope of this approach. Here a ¹H NMR method is described which allows structure determination of membrane peptides and small membrane proteins by ¹H 2D NMR in any type of non-deuterated detergent. The approach is based on regioselective excitation of protein resonances with DANTE-Z or spin-pinging pulse trains. It is shown that regioselective excitation of the amide-aromatic region of solubilized membrane proteins and peptides leads to an almost complete suppression of the two orders of magnitude higher contribution of the protonated detergent to the ¹H NMR spectrum. Consistently TOCSY, COSY and NOESY sequences incorporating such regioselective excitation in the F2 dimension yield protein ¹H 2D NMR spectra of quality comparable to those obtained in deuterated detergents. Regioselective TOCSY and NOESY spectra display all through-bond and through-space correlations within amide-aromatic protons and between these protons and aliphatic and -protons. Regioselective COSY spectra provide scalar coupling constants between amide and -protons. Application of the method to the membrane-active peptide mastoparan X, solubilized in n-octylglucoside, yields complete sequence-specific assignments and extensive secondary structure-related spatial proximities and coupling constants. It is shown that mastoparan adopts an -helical conformation when bound to nonionic detergent micelles. The present method is expected to increase the applicability of ¹H solution NMR methods to membrane proteins and peptides.Abbreviations 2D NMR two-dimensional NMR - COSY correlated spectroscopy - DANTE delays alternating nutations for tailored excitation - NOESY nuclear Overhauser enhancement spectroscopy - TOCSY total correlation spectroscopy     

3D structure of bovine pancreatic ribonuclease A in aqueous solution: An approach to tertiary structure determination from a small basis of1H NMR NOE correlations

Summary A method is proposed to generate initial structures in cases where the distance geometry method may fail, such as when the set of¹H NMR NOE-based distance constraints is small in relation to the size of the protein. The method introduces an initial correlation between the and backbone angles (based on empirical observations) which is relaxed in later stages of the calculation. The obtained initial structures are refined by well-established methods of energy minimization and restrained molecular dynamics. The method is applied to determine the solution structure of Ribonuclease A (124 residues) from a NOE basis consisting of 467 NOE cross-correlations (97 intra-residue, 206 sequential, 23 medium-range and 141 long-range) obtained at 360 MHz. The global shape and backbone overall fold of the eight final refined structures are close to those shown by the crystal structure. A meaningful difference in the positioning of the catalytically important His¹¹⁹ side chain in the solution and crystal structures has been detected.     

A novel approach to decoy set generation: designing a physical energy function having local minima with native structure characteristics

We suggest a new approach to the generation of candidate structures (decoys) for ab initio prediction of protein structures. Our method is based on random sampling of conformation space and subsequent local energy minimization. At the core of this approach lies the design of a novel type of energy function. This energy function has local minima with native structure characteristics and wide basins of attraction. The current work presents our motivation for deriving such an energy function and also tests the derived energy function.Our approach is novel in that it takes advantage of the inherently rough energy landscape of proteins, which is generally considered a major obstacle for protein structure prediction. When local minima have wide basins of attraction, the protein's conformation space can be greatly reduced by the convergence of large regions of the space into single points, namely the local minima corresponding to these funnels. We have implemented this concept by an iterative process. The potential is first used to generate decoy sets and then we study these sets of decoys to guide further development of the potential. A key feature of our potential is the use of cooperative multi-body interactions that mimic the role of the entropic and solvent contributions to the free energy.The validity and value of our approach is demonstrated by applying it to 14 diverse, small proteins. We show that, for these proteins, the size of conformation space is considerably reduced by the new energy function. In fact, the reduction is so substantial as to allow efficient conformational sampling. As a result we are able to find a significant number of near-native conformations in random searches performed with limited computational resources.     

Examining genetic structure in a bogus yucca moth: a sequential approach to phylogeography

Understanding the phylogeography of a species requires not only elucidating patterns of genetic structure among populations, but also identifying the possible evolutionary events creating that structure. The use of a single phylogeographic test or analysis, however, usually provides a picture of genetic structure without revealing the possible underlying evolutionary causes. We used current analytical techniques in a sequential approach to examine genetic structure and its underlying causes in the bogus yucca moth Prodoxus decipiens (Lepidoptera: Prodoxidae). Both historical biogeography and recent human transplantations of the moth's host plants provided a priori expectations of the pattern of genetic structure and its underlying causes. We evaluated these expectations by using a progression of phylogenetic, demographic, and population genetic analyses of mtDNA sequence data from 476 individuals distributed across 25 populations that encompassed the range of P. decipiens. The combination of these analyses revealed that much of the genetic structure has evolved more recently than suggested by historical biogeography, has been influenced by changes in demography, and can be best explained by long distance dispersal and isolation by distance. We suggest that performing a suite of analyses that focus on different temporal scales may be an effective approach to investigating the patterns and causes of genetic structure within species.     

A complete algorithm to resolve ambiguity for intersubunit NOE assignment in structure determination of symmetric homo-oligomers

Assignment of nuclear Overhauser effect (NOE) data is a key bottleneck in structure determination by NMR. NOE assignment resolves the ambiguity as to which pair of protons generated the observed NOE peaks, and thus should be restrained in structure determination. In the case of intersubunit NOEs in symmetric homo-oligomers, the ambiguity includes both the identities of the protons within a subunit, and the identities of the subunits to which they belong. This paper develops an algorithm for simultaneous intersubunit NOE assignment and C(n) symmetric homo-oligomeric structure determinations, given the subunit structure. By using a configuration space framework, our algorithm guarantees completeness, in that it identifies structures representing, to within a user-defined similarity level, every structure consistent with the available data (ambiguous or not). However, while our approach is complete in considering all conformations and assignments, it avoids explicit enumeration of the exponential number of combinations of possible assignments. Our algorithm can draw two types of conclusions not possible under previous methods: (1) that different assignments for an NOE would lead to different structural classes, or (2) that it is not necessary to uniquely assign an NOE, since it would have little impact on structural precision. We demonstrate on two test proteins that our method reduces the average number of possible assignments per NOE by a factor of 2.6 for MinE and 4.2 for CCMP. It results in high structural precision, reducing the average variance in atomic positions by factors of 1.5 and 3.6, respectively.     

GLASS: A tool to visualize protein structure prediction data in three dimensions and evaluate their consistency

When a protein sequence does not share any significant sequence similarity with a protein of known structure, homology modeling cannot be applied. However, many novel and interesting methods, such as secondary structure prediction, fold recognition, and prediction of long-range interactions, are being developed and have been shown to be reasonably successful in predicting protein structures from sequence data and evolutionary information. The a priori evaluation of the correctness of a prediction obtained by one of these methods is however often problematic. Consequently, it is important to use all available information provided by as many different methods as possible and all the available experimental data about the protein of interest, since the consistency of the results is indicative of the reliability of the prediction. Hence the need has arisen for suitable tools able to compare results provided by different methods and evaluate their consistency. We have therefore constructed GLASS, a general platform to read, visualize, compare, and evaluate prediction results from many different sources and to project these prediction results into three dimensions. In addition, GLASS allows the comparison of selected parameters calculated for a model with the distribution observed in real protein structures, thus providing an easy way to test new methods for evaluating the likelihood of different structural models. GLASS can be considered as a “workbench” for structural predictions useful to both experimentalists and theoreticians. Proteins 30:339–351, 1998. © 1998 Wiley-Liss, Inc.     

Feeling like a burden to others and the wish to hasten death in patients with advanced illness: A systematic review

Studies that have explored the wish to hasten death (WTHD) in patients with advanced illness have found that the feeling of being a burden may trigger WTHD. Research suggests that both the feeling and the wish are indicators of multidimensional suffering whose meaning may depend on the patient's biographical background. Therefore, we carried out a systematic review and meta‐ethnography. Fourteen qualitative studies, reported in 16 articles, met the inclusion criteria. The analysis identified two themes: the personal and social dimensions that could help to explain the feeling of being a burden in these patients. These dimensions reveal how this feeling is linked to physical, psychological/emotional, existential and social factors. The feeling of being a burden cannot be understood without considering patients’ personal interpretation of their dependency or care needs, and hence it is also necessary to understand their biographical background. Such an understanding is crucial to inform clinical policies based on the moral duty to provide all patients with humane care.     

鸟类交通致死原因的种间行为差异分析(英文)

鸟类和其他动物经常死于车撞, 每年因车撞而死亡的个体可达数百万只。为什么有的物种比其他物种更易于死于车撞的问题,迄今未研究过。该文推测物种本身所具有的冒险行为可能是这些物种的某些个体更常死于车撞的一个原因。此外,不同物种个体在公路上的行为、个体数量、栖息地偏好、繁殖社会性以及健康状况都可能是影响个体车撞风险的潜在因素。2001—2006 年,对丹麦一个农村地区进行定期统计取样, 得到该地区不同种鸟类个体被车撞死事件发生的频率,以及周边环境中 50 种鸟类的个体数量数据, 并利用这些数据来检验我们的预测。车撞频率随鸟类个体数量线性增加和位于道路上或者低空飞越道路的个体所占比例增加,这两个因素几乎无法解释鸟类车撞频率差异。在考虑到丰富度这个因素后,发现那些鸟类惊飞距离短、面对潜在危险而更具有冒险性的物种个体更易遭遇车撞而亡。另外, 独居的物种、疟原虫感染率高的物种及因体型大而拥有大法氏囊的物种个体死于车撞的频率更高。这些发现表明, 一系列反映冒险行为、视敏度和健康状况的因子导致某些鸟类物种易受到车撞影响。