Quantitative comparison of experimental and simulated NOE intensities: Correlation with accuracy of oligonucleotide structure determination

Summary The relation between the match of experimental and simulated NOE intensities with the accuracy of structure determination of oligonucleotides has been investigated. A hypothetical experimental spectrum of the oligonucleotide d(CCAACGTTGG) from its known X-ray crystallographic structure (Privé, G.G. et al. (1991)J. Mol. Biol.,217, 177–199) has been generated with simplifying assumptions of single correlation time, leakage rate etc., and this spectrum has been simulated imposing various constraints in a manner as one would do in a real case. The hypothetical spectrum represents the case of an infinitely good experimental spectrum and therefore the study of quality of fit against quality of structure represents the limiting case of real situations. It has been shown that even with a limited number of NOEs, it is possible to approach the correct structure of the molecule by demanding a highly accurate fit between experimental and simulated NOE intensities. Distance geometry calculations have been used to probe the extent of structural degeneracies in the NOE intensity matching exercise.     

Automated combined assignment of NOESY spectra and three-dimensional protein structure determination

A procedure for automated protein structure determination is presented that is based on an iterative procedure during which the NOESY peak list assignment and the structure calculation are performed simultaneously. The input consists of a list of NOESY peak positions and a list of chemical shifts as obtained from sequence-specific resonance assignment. For the present applications of this approach the previously introduced NOAH routine was implemented in the distance geometry program DIANA. As an illustration, experimental 2D and 3D NOESY cross-peak lists of six proteins have been analyzed, for which complete sequence-specific ¹H assignments are available for the polypeptide backbone and the amino acid side chains. The automated method assigned 70–90% of all NOESY cross peaks, which is on average 10% less than with the interactive approach, and only between 0.8% and 2.4% of the automatically assigned peaks had a different assignment than in the corresponding manually assigned peak lists. The structures obtained with NOAH/DIANA are in close agreement with those from manually assigned peak lists, and with both approaches the residual constraint violations correspond to high-quality NMR structure determinations. Systematic comparisons of the bundles of conformers that represent corresponding automatically and interactively determined structures document the absence of significant bias in either approach, indicating that an important step has been made towards automation of structure determination from NMR spectra.     

Variability in automated assignment of NOESY spectra and three-dimensional structure determination: A test case on three small disulfide-bonded proteins

Three independent runs of automatic assignment and structure calculations were performed on three small proteins, calcicludine from the venom of the green mamba Dendroaspis angusticeps, -conotoxin PVIIA from the purple cone Conus purpurascens and HsTX1, a short scorpion toxin from the venom of Heterometrus spinnifer. At the end of all the runs, the number of cross peaks which remained unassigned (0.6%, 1.4% and 2% for calcicludine, -conotoxin and HsTX1, respectively), as well as the number of constraints which were rejected as producing systematic violations (2.7%, 1.0%, and 1.4% for calcicludine, -conotoxin and HsTX1, respectively) were low. The conformation of the initial model used in the procedure (linear model or constructed by homology) has no influence on the final structures. Mainly two parameters control the procedure: the chemical shift tolerance and the cut-off distance. Independent runs of structure calculations, using the same parameters, yield structures for which the rmsd between averaged structures and the rmsd around each averaged structure were of the same order of magnitude. A different cut-off distance and a different chemical shift tolerance yield rmsd values on final average structures which did not differ more than 0.5 Å compared to the rmsd obtained around the averaged structure for each calculation. These results show that the procedure is robust when applied to such a small disulfide-bonded protein.     

Interproton distance bounds from 2D NOE intensities: Effect of experimental noise and peak integration errors

Summary The effect of experimental and integration errors on the calculations in interproton distances from NOE intensities is examined. It is shown that NOE intensity errors can have a large impact on the distances determined. When multiple spin (spin diffusion) effects are significant, the calculated distances are often underestimated, even when using a complete relaxation matrix analysis. In this case, the bias of distances to smaller values is due to the random errors in the NOE intensities. We show here that accurate upper and lower bounds of the distances can be obtained if the intensity errors are properly accounted for in the complete relaxation matrix calculations, specifically the MARDIGRAS algorithm. The basic MARDIGRAS algorithm has been previously described [Borgias, B.A. and James, T.L. (1990) J. Magn. Reson., 87, 475–487]. It has been shown to provide reasonably good interproton distance bounds, but experimental errors can compromise the quality of the resulting restraints, especially for weak cross peaks. In a new approach introduced here, termed RANDMARDI (random error MARDIGRAS), errors due to random noise and integration errors are mimicked by the addition of random numbers from within a specified range to each input intensity. Interproton distances are then calculated for the modified intensity set using MARDIGRAS. The distribution of distances that define the upper and lower distance bounds is obtained by using N randomly modified intensity sets. RANDMARDI has been used in the solution structure determination of the interstrand cross-link (XL) formed between 4-hydroxymethyl-4,5,8-trimethylpsoralen (HMT) and the DNA oligomer d(5-GCGTACGC-3)₂ [Spielmann, H.P. et al. (1995) Biochemistry, 34, 12937–12953]. RANDMARDI generates accurate distance bounds from the experimental NOESY cross-peak intensities for the fixed (known) interproton distances in XL. This provides an independent internal check for the ability of RANDMARDI to accurately fit the experimental data. The XL structure determined using RANDMARDI-generated restrains is in good agreement with other biophysical data that indicate that there is no bend introduced into the DNA by the cross-link. In contrast, isolated spin-pair approximation calculations give distance restraints that, when applied in a restrained molecular dynamics protocol, produce a bent structure.Abbreviations NOE nuclear Overhauser effect - SD standard deviation - HMT 4-hydroxymethyl-4,5,8-trimethylpsoralen - XL psoralen-DNA interstrand cross-link     

Automated assignment and 3D structure calculations using combinations of 2D homonuclear and 3D heteronuclear NOESY spectra

The NOAH/DIAMOD suite uses feedback filtering and self-correcting distance geometry to generate 3D structures from unassigned NOESY spectra. In this study we determined the minimum set of experiments needed to generate a high quality structure bundle. Different combinations of 3D ¹⁵N-edited, ¹³C-edited HSQC-NOESY and 2D homonuclear ¹H-¹H NOESY spectra of the 77 amino acid protein, myeloid progenitor inhibitory factor-1 (MPIF-1) were used as input for NOAH/DIAMOD calculations. The quality of the assignments of NOESY cross peaks and the accuracy of the automatically generated 3D structures were compared to those obtained with a conventional manual procedure. Combining data from two types of experiments synergistically increased the number of peaks assigned unambiguously in both individual spectra. As a general trend for the accuracy of the structures we observed structural variations in the backbone fold of the final structures of about 2 Å for single spectral data, of 1 Å to 1.5 Å for double spectral data, and of 0.6 Å for triple spectral data sets. The quality of the assignments and 3D structures from the optimal data using all three spectra were similar to those obtained from traditional assignment methods with structural variations within the bundle of 0.6 Å and 1.3 Å for backbone and heavy atoms, respectively. Almost all constraints (97%) of the automatic NOESY cross peak assignments were cross compatible with the structures from the conventional manual assignment procedure, and an even larger proportion (99%) of the manually derived constraints were compatible with the automatically determined 3D structures. The two mean structures determined by both methods differed only by 1.3 Å rmsd for the backbone atoms in the well-defined regions of the protein. Thus NOAD/DIAMOD analysis of spectra from labeled proteins provides a reliable method for high throughput analysis of genomic targets.     

Population structure in the Connecticut Valley: II. A comparison of multidimensional scaling solutions of migration matrices and isonymy

In a previous paper (Swedlund et al., 1984) we have described the population structure of the historical Connecticut River Valley of Massachusetts in terms of matrimonial migration matrices. Using procedures described by Morton (1973), Harpending and Jenkins (1974), Jorde (1980), and others the exchanges between subdivisions which make up the matrices are made column stochastic and analyzed to predict genetic kinship. Subsequently the kinship estimates within and between subdivisions can be interpreted as genetic covariance and compared to the actual geographic distances between the respective subdivisions using a principal components analysis. In the present paper we extend these results by applying nonmetric multidimensional scaling to the migration matrices, and to isonymy matrices based on the same communities. We demonstrate that the multidimensional scaling configurations of marital migration represent the actual geographic relationships between the communities quite effectively for this particular case study from historical Massachusetts. Moreover, we argue that while these migration data may provide good estimates of social and genetic exchange between the subdivisions, surname analysis may also be informative of processes not revealed in the migration matrices alone.     

Improved simulation of NOESY spectra by RELAX-JT2 including effects of J-coupling, transverse relaxation and chemical shift anisotropy

RELAX-JT2 is an extension of RELAX, a program for the simulation of 1H 2D NOESY spectra and (15)N or (13)C edited 3D NOESY-HSQC spectra of biological macromolecules. In addition to the already existing NOE-simulation it allows the proper simulation of line shapes by the integrated calculation of T(2) times and multiplet structures caused by J-couplings. Additionally the effects of relaxation mediated by chemical shift anisotropy are taken into account. The new routines have been implemented in the program AUREMOL, which aims at the automated NMR structure determination of proteins in solution. For a manual or automatic assignment of experimental spectra that is based on the comparison with the corresponding simulated spectra, the additional line shape information now available is a valuable aid. The new features have been successfully tested with the histidine-containing phosphocarrier protein HPr from Staphylococcus carnosus.     

Hyperolactone C: determination of its absolute configuration by comparison of experimental and calculated CD spectra

A detailed conformational analysis of hyperolactone C diastereomers and enantiomers ((5R,9R),(5S,9S) and (5S,9R),(5R,9S)) was done with molecular mechanics and density functional theory methods. Time-dependent density functional theory (B3PW91/TZVP) was used to calculate electronic transition energies (UV/vis spectra) and rotational strengths of the respective conformations. The effect of solvation (acetonitrile solution) on excitation energies and electronic circular dichroism was approximated by the polarizable continuum model. By comparison of the simulated CD spectrum with that measured for hyperolactone C isolated from Hypericum lloydii, its absolute configuration can be assigned as (5S,9S).     

New applications of 2D filtered/edited NOESY for assignment and structure elucidation of RNA and RNA-protein complexes

NMR spectra of large RNAs are difficult to assign because of extensive spectral overlap and unfavorable relaxation properties. Here we present a new approach to facilitate assignment of RNA spectra using a suite of four 2D-filtered/edited NOESY experiments in combination with base-type-specific isotopically labeled RNA. The filtering method was developed for use in 3D filtered NOESY experiments (Zwahlen et al., 1997), but the 2D versions are both more sensitive and easier to interpret for larger RNAs than their 3D counterparts. These experiments are also useful for identifying intermolecular NOEs in RNA-protein complexes. Applications to NOE assignment of larger RNAs and an RNA-protein complex are presented.     

Filtering methods for selection of singlet and doublet signals in NMR spectra of DNA oligomers

Summary The base proton (purine H8 and pyrimidine H6) resonances are key signals for the assignment of the proton resonances of DNA oligomers. They are classified into two groups, i.e., cytosine H6 signals, observed as doublets, and the other base proton signals, observed as singlets. Here we propose some experiments for distinguishing the cytosine H6 signals from the other base proton signals. Moreover, the ability of signal selection and the sensitivity as to signal detection were compared for all experiments, and the optimum conditions for spectral measurements were surveyed. Some of the experiments were employed as the NOESY detection pulse. Previously proposed experiments, such as HOENOE and HAL, were also used in the comparison.     

Circular dichroism spectra of twelve short DNA restriction fragments of known sequence: a comparison of measured and calculated spectra.

The CD spectra of twelve DNA restriction fragments ranging in size from 12 to 360 base pairs are reported. Since the sequences of these fragments are known, it is possible to calculate their CD spectra from a set of nearest neighbor contributions derived from a combination of synthetic polydeoxyribonucleotides. While the calculations lead to good agreement in the negative band at approximately 245 nm, they generally reproduce the positive band at approximately 270 nm only poorly. The experimentally observed positive band consists of two peaks centered around 270 and 285 nm. The comparison of calculated and measured spectra reveals that end effects lead to increased disagreement for fragments smaller than approximately 40 base pairs. The disagreement between calculated and measured spectra can be partially attributed to the fraction of next nearest neighbors in the DNAs, which are also in the spectral components. Thus, the sequence specific CD contributions in the long wavelength region of the spectra extend at least to next nearest neighbor nucleotides and may extend beyond.     

MUFOLD: A new solution for protein 3D structure prediction

There have been steady improvements in protein structure prediction during the past 2 decades. However, current methods are still far from consistently predicting structural models accurately with computing power accessible to common users. Toward achieving more accurate and efficient structure prediction, we developed a number of novel methods and integrated them into a software package, MUFOLD. First, a systematic protocol was developed to identify useful templates and fragments from Protein Data Bank for a given target protein. Then, an efficient process was applied for iterative coarse‐grain model generation and evaluation at the Cα or backbone level. In this process, we construct models using interresidue spatial restraints derived from alignments by multidimensional scaling, evaluate and select models through clustering and static scoring functions, and iteratively improve the selected models by integrating spatial restraints and previous models. Finally, the full‐atom models were evaluated using molecular dynamics simulations based on structural changes under simulated heating. We have continuously improved the performance of MUFOLD by using a benchmark of 200 proteins from the Astral database, where no template with >25% sequence identity to any target protein is included. The average root‐mean‐square deviation of the best models from the native structures is 4.28 Å, which shows significant and systematic improvement over our previous methods. The computing time of MUFOLD is much shorter than many other tools, such as Rosetta. MUFOLD demonstrated some success in the 2008 community‐wide experiment for protein structure prediction CASP8. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

A procedure for large-scale isolation of RNA-free plasmid and phage DNA without the use of RNase

A preparative procedure for the large-scale isolation of plasmid DNA without the use of RNAse is described. Crude plasmid DNA is prepared using a standard boiling method. High-molecular-weight RNA is removed by precipitation with LiCl, and low-molecular-weight RNA is removed by sedimentation through high-salt solution. The procedure is inexpensive, rapid, simple, and particularly suitable for processing several large-scale preparations simultaneously. A similar procedure has been developed for preparation of lambda-phage DNA.     

DNA extractions from deep subseafloor sediments: novel cryogenic-mill-based procedure and comparison to existing protocols

Extracting DNA from deep subsurface sediments is challenging given the complexity of sediments types, low biomasses, resting structures (spores, cysts) frequently encountered in deep sediments, and the potential presence of enzymatic inhibitors. Promising results for cell lysis efficiency were recently obtained by use of a cryogenic mill (Lipp et al., 2008). These findings encouraged us to devise a DNA extraction protocol using this tool. Thirteen procedures involving a combination of grinding in liquid nitrogen (for various durations and beating rates) with different chemical solutions (phenol, chloroform, SDS, sarkosyl, proteinase, GTC), or with use of DNA recovery kits (MagExtractor®) were compared. Effective DNA extraction was evaluated in terms of cell lysis efficiency, DNA extraction efficiency, DNA yield and determination of prokaryotic diversity. Results were compared to those obtained by standard protocols: the FastDNA®SPIN kit for soil and the Zhou protocol. For most sediment types grinding in a cryogenic mill at a low beating rate in combination with direct phenol-chloroform extraction resulted in much higher DNA yields than those obtained using classical procedures. In general (except for clay-rich sediments), this procedure provided high-quality crude extracts for direct downstream nested-PCR, from cell numbers as low as 1.1 × 10⁶ cells/cm³. This procedure is simple, rapid, low-cost, and could be used with minor modifications for large-scale DNA extractions for a variety of experimental goals.     

A rapid,cost-effective procedure for the extraction of microbial DNA from soil

Here we describe a DNA extraction method that is based on a simple, rapid polyvinylpolypyrrolidone–calcium chloride precipitation to release microorganisms from the soil combined with lysozyme–proteinase–SDS lysis of the microbial community. The extracted DNA is of high quality and allows direct detection of specific genes by the polymerase chain reaction (PCR) as well as cloning of indigenous microbial DNA. This method facilitates the extraction of 36 500-mg soil samples simultaneously in a 2-h period by one person. The procedure is safe, inexpensive, and does not require specialized equipment or generate hazardous wastes.     

叶片和群落尺度净光合速率关系的探讨

叶片净光合速率(P_n)是研究光合作用机理的基本尺度; 而群落净光合速率(P_c)是研究群落光合能力及其与外部环境因子间关系的更好尺度, 特别是区域乃至全球尺度碳循环的研究, 需要将叶片尺度的生理生态模型扩展到冠层尺度。理论上, 群落内所有叶片的累积P_n与实测群落净气体交换速率(NCE)是相等的, 但在野外实际观测中, 两者之间的相互关系目前尚未见报道。该文选取敖汉苜蓿(Medicago sativa ‘Aohan’)人工草地, 采用美国LI-COR公司生产的便携式光合测定系统LI-6400测定P_n, 结合叶面积指数等参数推算P_c, 利用LI-8100连接同化箱测定生态系统净气体交换速率(NEE), 加上土壤呼吸速率, 得到NCE。结果表明: P_c为3.52 μmol CO₂·m^-2·s^-1, 与实测NCE (3.56 μmol CO₂·m^-2·s^-1)基本相等。这表明: 可利用P_n, 结合叶面积指数、群落叶片数目、健康叶片比例和群落可接收有效光照的平均比例等4个关键参数, 准确地换算P_c。然而, 利用同化箱式法测定群落呼吸速率时, 不可避免地会包含土壤呼吸, 所以在观测NCE时, 需要同时测定土壤呼吸。此外, 在冠层模型中, 群落垂直结构和光量子的非线性响应不可忽视。     

A procedure for the quantitation of relaxed closed circular DNA in the presence of superhelical DNA: an improved fluorometric assay for nicking-closing enzyme.

The procedure described here takes advantage of the recently discovered single-strand-specific endonuclease activity of snake-venom phosphodiesterase to convert supercoiled PM2 DNA (DNA I′), but not relaxed DNA (DNA I′), to open forms of DNA. The DNA I' was quantitated using a fiuorometric method for covalently closed circular DNA (A. R. Morgan and D. E. Pulleyblank, 1974, Biochem. Biophys. Res. Commun.61, 396–403). The percentage of DNA I′ in mixtures of DNAs I and I′ can be determined to ±l%. The procedure was used as an assay for a nicking-closing enzyme activity partially purified from simian virus 40-infected monkey cells. The assay is linear from 0 to 0.4 μg DNA I′ produced and reproducible to ±0.01 μg DNA I′.     

A note on sequence-dependence of DNA structure

A circular dichroism study was conducted on the solution structure of several different oligonucleotides, whose X-ray structures have been solved. It is suggested that in aqueous solution the oligonucleotides can form structures that maintain geometrical elements which are typical of B-DNA, A-DNA, and their intermediate forms. It is shown that 5'GGATGGGAG:5'CTCCCATCC, which forms an A-DNA helix in the crystal state (McCall et al. 1986), in aqueous solution maintains an A-DNA like structure at temperatures below 10 degrees C. At temperatures between 10 degrees C and 25 degrees C it shows a tendency to form an intermediate structure between A-DNA and B-DNA. Also, it is shown that TFE does not cause a transition from B-DNA to A-DNA helix in short DNA fragments, but instead disrupts the helix.