Quantitative evaluation of experimental NMR restraints (original) (raw)
Concepts and tools for NMR restraint analysis and validation
Concepts in Magnetic Resonance, 2004
The quality of NMR-derived biomolecular structure models can be assessed by validation on the level of structural characteristics as well as the NMR data used to derive the structure models. Here, an overview is given of the common methods to validate experimental NMR data. These methods provide measures of quality and goodness of fit of the structure to the data. A detailed discussion is given of newly developed methods to assess the information contained in experimental NMR restraints, which provide powerful tools for validation and error analysis in NMR structure determination.
Extending the Applicability of Exact Nuclear Overhauser Enhancements to Large Proteins and RNA
Chembiochem : a European journal of chemical biology, 2018
Distance-dependent NOEs are one of the most popular and important experimental restraints for calculating NMR structures. Despite this, they are mostly employed as semi-quantitative upper distance bounds, which discards a wealth of information that is encoded in the cross-relaxation rate constant. Information that is lost includes exact distances between protons and dynamics that occur on the sub-millisecond time-scale. Our recently introduced exact measurement of the NOE (eNOE) requires little additional experimental effort relative to other NMR observables. So far, we have used eNOEs to calculate multi-state ensembles of proteins up to ~150 residues. Here, we briefly revisit the eNOE methodology and present two new directions for the use of eNOEs: Applications to large proteins and RNA.
Journal of Bioinformatics and Computational Biology, 2008
Nuclear Overhauser effects (NOE) distance constraints and torsion angle constraints are major conformational constraints for nuclear magnetic resonance (NMR) structure refinement. In particular, the number of NOE constraints has been considered as an important determinant for the quality of NMR structures. Of course, the availability of torsion angle constraints is also critical for the formation of correct local conformations. In our recent work, we have shown how a set of knowledge-based short-range distance constraints can also be utilized for NMR structure refinement, as a complementary set of conformational constraints to the NOE and torsion angle constraints. In this paper, we show the results from a series of structure refinement experiments by using different types of conformational constraints — NOE, torsion angle, or knowledge-based constraints — or their combinations, and make a quantitative assessment on how the experimentally acquired constraints contribute to the quali...
Trends in Food Science & Technology, 2019
The characterization of the original chemical structure and induced changes of micro-and macro-molecules using analytical techniques with concise and detailed outcomes is potentially one of the major challenges for food scientists. To this end, the non-invasive nuclear magnetic resonance (NMR) technique can play a significant role through employment of different NMR methods. The Nuclear Overhauser effect (NOE) and rotating-frame Overhauser effect (ROE) techniques are powerful NMR methods that have attracted great interest because they provide precise information about the three dimensional spatial structure of the molecules, as well as about possible chemical reactions and interactions. Scope and approach In this article, we reviewed the basic principles as well as applications of two NMR techniques: Nuclear Overhauser effect spectroscopy (NOESY) and rotating-frame Overhauser effect spectroscopy (ROESY). Hereby, we focused mainly on the applications and importance of these techniques in food science research. Both the structural (configuration and conformation) changes and the complexes formed by interacting compounds could be better studied using these techniques. Key findings and conclusions The inter-and intra-molecular interactions within food-based ingredient mixtures, as well as configurational and conformational analyses can be more efficiently studied with the aid of NOESY and ROESY. These methods as complementary analysis tools can be exploited for the straightforward elucidation of the spatial proximity of either novel, native or modified compounds. In the future, these techniques may be helpful to better understand the interaction between polymers, such as protein-polysaccharide interactions.
Model-independent refinement of interproton distances generated from 1H NMR overhauser intensities
Journal of Magnetic Resonance (1969), 1991
A recursive method to refine interproton distances compatible with two-dimensional nuclear Overhauser effect (NOESY) experiments has been tested. Convergence does not depend on the initial estimate of the parameters. Hence, no approximate initial structure of the molecule is required: the iterative process can be started from the experimentally measured NOESY cross-peak volumes, supplemented with arbitrary cross-peak and autopeak values to obtain an initial NOESY matrix. The relaxation matrix is calculated from the NOESY matrix, and its diagonal elements (p,) are adjusted at each iteration until the difference between theoretical and experimental cross peaks is a minimum. The improvement comes from using interproton distances calculated from the off-diagonal (uU) elements to generate p, values. The method was applied to alumichrome, a rigid cyclohexapeptide of virtually identical solution and crystallographic structures. The experimental data consisted of the integrated volumes of NOESY cross peaks at 500 MHz. Convergence was tested by resorting to different initial conditions, one of them being a NOESY matrix in which the experimentally unobserved off-diagonal elements were set equal to zero and the diagonal elements to 0.5. The iterations rapidly converge, in all cases, to a set of distances whose root-mean-squares deviation (rmsd) from the crystallographic distances is co.05 A. The acronym MIDGE (model-independent distance generation) for the procedure is proposed.
The Exact Nuclear Overhauser Enhancement: Recent Advances
Molecules
Although often depicted as rigid structures, proteins are highly dynamic systems, whose motions are essential to their functions. Despite this, it is difficult to investigate protein dynamics due to the rapid timescale at which they sample their conformational space, leading most NMR-determined structures to represent only an averaged snapshot of the dynamic picture. While NMR relaxation measurements can help to determine local dynamics, it is difficult to detect translational or concerted motion, and only recently have significant advances been made to make it possible to acquire a more holistic representation of the dynamics and structural landscapes of proteins. Here, we briefly revisit our most recent progress in the theory and use of exact nuclear Overhauser enhancements (eNOEs) for the calculation of structural ensembles that describe their conformational space. New developments are primarily targeted at increasing the number and improving the quality of extracted eNOE distance restraints, such that the multi-state structure calculation can be applied to proteins of higher molecular weights. We then review the implications of the exact NOE to the protein dynamics and function of cyclophilin A and the WW domain of Pin1, and finally discuss our current research and future directions.
Journal of Magnetic Resonance (1969), 1988
COMATOSE (complete matrix analysis torsion optimized structure) is a structurerefinement program based on the quantitative calculation of 2D NOE intensities taking into account the effects of network relaxation and spin diffusion (J. W. Keepers and T. L. James, J. Magn. Reson. 57, 404 (1984)). The macromolecular structure is defined by fixed bondlengths and two-bond angles, leaving only torsion angles and residue orientations as variable parameters. The performance of this structure refinement algorithm with idealized and experimental 2D NOE intensities for oligonucleotides is discussed. The structural information available from COMATOSE is compared with the distances obtained by analysis of the 2D NOE intensities, and with the distances available from the direct solution of the intensity eigenvalue problem. The most commonly used calculation of distances relies on the assumption that the intensities can be approximated by assuming that the individual spin pairs are essentially isolated from all other protons. This approach systematically results in underestimation of distances. Direct solution of the eigenvalue problem when all intensities (i.e., relaxation pathways) are not accounted for results in large errors especially at longer distances (greater than-3 A). COMATOSE is capable of optimizing the structure in favorable cases and, at least, yielding a set of reliable proton-proton distances (relative error-10%). In most practical applications, an iterative approach to the development of the structure should probably IX taken. An initial trial structure is subjected to COMATOSE to obtain accurate distance estimates from the 2D NOE. These in turn can be used as constraints in energy minimization of the structure to arrive at an improved structure for recycling into COMATOSE. o 1988 Academic Press, Inc.
Journal of the American Chemical Society, 2004
The major rate-limiting step in high-throughput NMR protein structure determination involves the calculation of a reliable initial fold, the elimination of incorrect nuclear Overhauser enhancement (NOE) assignments, and the resolution of NOE assignment ambiguities. We present a robust approach to automatically calculate structures with a backbone coordinate accuracy of 1.0-1.5 Å from datasets in which as much as 80% of the long-range NOE information (i.e., between residues separated by more than five positions in the sequence) is incorrect. The current algorithm differs from previously published methods in that it has been expressly designed to ensure that the results from successive cycles are not biased by the global fold of structures generated in preceding cycles. Consequently, the method is highly error tolerant and is not easily funnelled down an incorrect path in either three-dimensional structure or NOE assignment space. The algorithm incorporates three main features: a linear energy function representation of the NOE restraints to allow maximization of the number of simultaneously satisfied restraints during the course of simulated annealing; a method for handling the presence of multiple possible assignments for each NOE cross-peak which avoids local minima by treating each possible assignment as if it were an independent restraint; and a probabilistic method to permit both inactivation and reactivation of all NOE restraints on the fly during the course of simulated annealing. NOE restraints are never removed permanently, thereby significantly reducing the likelihood of becoming trapped in a false minimum of NOE assignment space. The effectiveness of the algorithm is demonstrated using completely automatically peak-picked experimental NOE data from two proteins: interleukin-4 (136 residues) and cyanovirin-N (101 residues). The limits of the method are explored using simulated data on the 56-residue B1 domain of Streptococcal protein G.
Molecular dynamics and accuracy of NMR structures: Effects of error bounds and data removal
Proteins: Structure, Function, and Genetics, 1999
The effect of internal dynamics on the accuracy of nuclear magnetic resonance (NMR) structures was studied in detail using model distance restraint sets (DRS) generated from a 6.6 nanosecond molecular dynamics trajectory of bovine pancreatic trypsin inhibitor. The model data included the effects of internal dynamics in a very realistic way. Structure calculations using different error estimates were performed with iterative removal of systematically violated restraints. The accuracy of each calculated structure was measured as the atomic root mean square (RMS) difference to the optimized average structure derived from the trajectory by structure factors refinement. Many of the distance restraints were derived from NOEs that were significantly affected by internal dynamics. Depending on the error bounds used, these distance restraints seriously distorted the structure, leading to deviations from the coordinate average of the dynamics trajectory even in rigid regions. Increasing error bounds uniformly for all distance restraints relieved the strain on the structures. However, the accuracy did not improve. Significant improvement of accuracy was obtained by identifying inconsistent restraints with violation analysis, and excluding them from the calculation. The highest accuracy was obtained by setting bounds rather tightly, and removing about a third of the restraints. The limiting accuracy for all backbone atoms was between 0.6 and 0.7 Å. Also, the precision of the structures increased with removal of inconsistent restraints, indicating that a high precision is not simply the consequence of tight error bounds but of the consistency of the DRS. The precision consistently overestimated the accuracy. Proteins 1999;34:453-463. 1999 Wiley-Liss, Inc.
Carbohydrate Research, 1992
The conformational preference of the disaccharide a-L-Rhap-(1 -$2)-cY-L-Rhap-(l + OMe) (11 about the glycosidic torsion angles, I$ and ,wasstudiedbyNMRNOESYspectroscopyandmolecularmechanicscalculations.TheNOEdatawereconsistentwitheitheroftwodistinctconformationsclosetominimaonacalculated4/, was studied by NMR NOESY spectroscopy and molecular mechanics calculations. The NOE data were consistent with either of two distinct conformations close to minima on a calculated 4/,wasstudiedbyNMRNOESYspectroscopyandmolecularmechanicscalculations.TheNOEdatawereconsistentwitheitheroftwodistinctconformationsclosetominimaonacalculated4/ potential energy surface. Starting from the lowest energy conformation, a l-ns molecular dynamics (MD) trajectory was computed in vacua, from which the NOE curves were simulated and compared to the experimentally observed NOESY data. 0008-6215/92/$05.00 0 1992 -Elsevier Science Publishers B.V. All rights reserved 196 G. Widmalm et al. / Carbohydr. Res. 229 (1992) 195-211 disaccharide L-w-L-Rhap-(1 + 2)-a-L-Rhap-(1 + OMe) (1) based on NOESY and molecular mechanics studies. EXPERIMENTAL Disaccharide nomenclature conventions .-Atoms in the glycosyl group are assigned primed numbers; those in the methyl glycoside are unprimed. The glycosidic torsion angle 4 is defined by the atoms 0-5'-C-l'-O-2-C-2
The Exact Nuclear Overhauser Enhancement
2018
Although often depicted as rigid structures, proteins are highly dynamic systems, whose motions are essential to their functions. Despite this, it is difficult to investigate protein dynamics due to the rapid timescale at which they sample their conformational space, leading most NMR-determined structures to represent only an averaged snapshot of the dynamic picture. While NMR relaxation measurements can help to determine local dynamics, it is difficult to detect translational or concerted motion, and only recently have significant advances been made to make it possible to acquire a more holistic representation of the dynamics and structural landscapes of proteins. Here, we briefly revisit our most recent progress in the theory and use of exact nuclear Overhauser enhancements (eNOEs) for the calculation of structural ensembles that describe their conformational space. New developments are primarily targeted at increasing the number and improving the quality of extracted eNOE distanc...
Combining NMR and EPR Methods for Homodimer Protein Structure Determination
2010
There is a general need to develop more powerful and more robust methods for structural characterization of homodimers, homo-oligomers, and multiprotein complexes using solution-state NMR methods. In recent years, there has been increasing emphasis on integrating distinct and complementary methodologies for structure determination of multiprotein complexes. One approach not yet widely used is to obtain intermediate and long-range distance constraints from paramagnetic relaxation enhancements (PRE) and electron paramagnetic resonance (EPR)-based techniques such as double electron electron resonance (DEER), which, when used together, can provide supplemental distance constraints spanning to 10-70 Å. In this Communication, we describe integration of PRE and DEER data with conventional solution-state nuclear magnetic resonance (NMR) methods for structure determination of Dsy0195, a homodimer (62 amino acids per monomer) from Desulfitobacterium hafniense. Our results indicate that combination of conventional NMR restraints with only one or a few DEER distance constraints and a small number of PRE constraints is sufficient for the automatic NMR-based structure determination program CYANA to build a network of interchain nuclear Overhauser effect constraints that can be used to accurately define both the homodimer interface and the global homodimer structure. The use of DEER distances as a source of supplemental constraints as described here has virtually no upper molecular weight limit, and utilization of the PRE constraints is limited only by the ability to make accurate assignments of the protein amide proton and nitrogen chemical shifts.
Blind Testing of Routine, Fully Automated Determination of Protein Structures from NMR Data
Structure, 2012
The protocols currently used for protein structure determination by nuclear magnetic resonance (NMR) depend on the determination of a large number of upper distance limits for proton-proton pairs. Typically, this task is performed manually by an experienced researcher rather than automatically by using a specific computer program. To assess whether it is indeed possible to generate in a fully automated manner NMR structures adequate for deposition in the Protein Data Bank, we gathered 10 experimental data sets with unassigned nuclear Overhauser effect spectroscopy (NOESY) peak lists for various proteins of unknown structure, computed structures for each of them using different, fully automatic programs, and compared the results to each other and to the manually solved reference structures that were not available at the time the data were provided. This constitutes a stringent ''blind'' assessment similar to the CASP and CAPRI initia-tives. This study demonstrates the feasibility of routine, fully automated protein structure determination by NMR.
An evaluation of detergents for NMR structural studies of membrane proteins
Journal of Biomolecular Nmr, 2004
Structural information on membrane proteins lags far behind that on soluble proteins, in large part due to difficulties producing homogeneous, stable, structurally relevant samples in a membrane-like environment. In this study 25 membrane mimetics were screened using 2D 1 H-15 N heteronuclear single quantum correlation NMR experiments to establish sample homogeneity and predict fitness for structure determination. A single detergent, 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)] (LPPG), yielded high quality NMR spectra with sample lifetimes greater than one month for the five proteins tested -R. sphaeroides LH1 α and β subunits, E. coli and B. pseudofirmus OF4 ATP synthase c subunits, and S. aureus small multidrug resistance transporter -with 1, 2, or 4 membrane spanning α-helices, respectively. Site-specific spin labeling established interhelical distances in the drug transporter and genetically fused dimers of c subunits in LPPG consistent with in vivo distances. Optical spectroscopy showed that LH1 β subunits form native-like complexes with bacteriochlorphyll a in LPPG. All the protein/micelle complexes were estimated to exceed 100 kDaltons by translational diffusion measurements. However, analysis of 15 N transverse, longitudinal and 15 N{ 1 H} nuclear Overhauser effect relaxation measurements yielded overall rotational correlation times of 8 to 12 nsec, similar to a 15-20 kDalton protein tumbling isotropically in solution, and consistent with the high quality NMR data observed.