How accurately can oligonucleotide structures be determined from the hybrid relaxation rate matrix/NOESY distance restrained molecular dynamics approach?

The accuracy and precision of structures derived from a combined hybrid relaxation rate matrix/NOESY distance restrained molecular dynamics methodology were examined with simulations that included typical experimental errors. NOESY data were simulated for a DNA dodecamer duplex, d-(CGCGAATTCGCG)2, with added volume error of approximately 20% and low-level thermal noise. Distances derived from a hybrid relaxation matrix analysis of the NOE data were used as constraints in molecular dynamics driven structural refinements of several initial model geometries. The final structures were compared against results obtained from the traditional isolated two-spin approximation treatment of these NOESY volumes and also against refined structures that employed error-free data. Results show that the structures derived from the relaxation rate matrix analysis of the NOESY data are more accurate than those derived from a simple two-spin approximation analysis and it is possible to achieve refinement to the level of simulated experimental error. Results may be significantly improved with the use of either more accurately measured NOESY volumes or additional matrix-derived constraints. Many of the helical parameters and backbone torsional angles may be accurately reproduced by the hybrid matrix methodology.     

Solution structure of the EcoRI DNA sequence: refinement of NMR-derived distance geometry structures by NOESY spectrum back-calculations

The solution structure of the self-complementary DNA duplex [d(CGCGAATTCGCG)]2, which contains the EcoRI restriction site sequence GAATTC at the center, has been studied by two-dimensional nuclear magnetic resonance spectroscopy. Time-dependent nuclear Overhauser effect spectra were used to obtain the initial cross-relaxation rates between 155 pairs of protons. These initial cross-relaxation rates were converted into interproton distances and entered into a distance (bounds) matrix. A distance geometry algorithm (DSPACE) was used to create embedded starting structures and to refine these structures until they showed good agreement with the distance matrix; symmetry constraints were included in the refinement procedure, making the two strands in the refined distance geometry structures virtually identical and significantly improving the agreement with the distance matrix. The NOESY spectrum for one of these distance geometry structures was then calculated from the explicit coordinates by numerically integrating all the z-magnetization transfer pathways among neighboring protons within a specified radius. Distances in this distance geometry structure that did not agree with the experimental NOESY time course were then adjusted accordingly. This process was iterated until a good agreement between calculated and experimental NOESY spectra was reached. The final structure, which generates good agreement with the experimental NOESY spectrum, displays kinks at the C3-G4 base step and at the A6-T7 base step that appear to be similar to those reported for the EcoRI restriction site DNA bound to its endonuclease. The solution structure is not the same as the crystal structure of this DNA duplex.     

Three-dimensional structure of acyl carrier protein determined by NMR pseudoenergy and distance geometry calculations

Distance constraints from two-dimensional NMR cross-relaxation data are used to derive a three-dimensional structure for acyl carrier protein from Escherichia coli. Several approaches to structure determination are explored. The most successful proves to be an approach that combines the early stages of a distance geometry program with energy minimization in the presence of NMR constraints represented as pseudopotentials. Approximately 450 proton to proton distance constraints including 50 long-range constraints were included in these programs. Starting structures were generated at random by the distance geometry program and energies minimized by a molecular mechanics module to give final structures. Seven of the structures were deemed acceptable on the basis of agreement with experimentally determined distances. Root-mean-square deviations from the mean of these structures for backbone atoms range from 2 to 3 A. All structures show three roughly parallel helices with hydrophobic residues facing inward and hydrophilic residues facing outward. A hydrophobic cleft is recognizable and is identified as a likely site for acyl chain binding.     

Solution structure of a LewisX analogue by off-resonance 1H NMR spectroscopy without use of an internal distance reference

Summary A recent ¹H NMR method has been applied to the determination of the solution structure and internal dynamics of a synthetic mixed C/O trisaccharide related to sialyl Lewis^x. Varying the rf field offset in ROESY-type experiments enabled the measurement of longitudinal and transverse dipolar cross-relaxation rates with high accuracy. Assuming that for each proton pair the motion could be represented by a single exponential autocorrelation function, it was possible to derive geometrical parameters (r) and dynamic parameters _cp. With this assumption, 224 cross-relaxation rates have been transformed into 30 interproton distance constraints and 30 dipolar correlation times. The distance constraints have been used in a simulated-annealing procedure. This trisaccharide exhibits a structure close to the O-glycosidic analogue, but its flexibility seems highly reduced. On the basis of the determined structure and dynamics, it is shown that no conformational exchange occurs, the molecule existing in the form of a unique family in aqueous solution. In order to assess the quality of the resulting structures and to validate this new experimental procedure of distance extraction, we finally compare these solution structures to the ones obtained using three different sets of distances deduced from three choices of internal reference. It appears that this procedure allows the determination of the most precise and accurate solution.Abbreviations COSY correlation spectroscopy - NOE nuclear Overhauser enhancement - NOESY nuclear Overhauser enhancement spectroscopy; rmsd, root-mean-square deviation - ROESY rotating frame Overhauser enhancement spectroscopy - SLe^x sialyl Lewis^x - TOCSY total correlation spectroscopy     

Determination of the complete three-dimensional structure of the trypsin inhibitor from squash seeds in aqueous solution by nuclear magnetic resonance and a combination of distance geometry and dynamical simulated annealing

The complete three-dimensional structure of the trypsin inhibitor from seeds of the squash Cucurbita maxima in aqueous solution was determined on the basis of 324 interproton distance constraints, 80 non-nuclear Overhauser effect distances, and 22 hydrogen-bonding constraints, supplemented by 27 phi backbone angle constraints derived from nuclear magnetic resonance measurements. The nuclear magnetic resonance input data were converted to the distance constraints in a semiquantitative manner after a sequence specific assignment of 1H spectra was obtained using two-dimensional nuclear magnetic resonance techniques. Stereospecific assignments were obtained for 17 of the 48 prochiral centers of the squash trypsin inhibitor using the floating chirality assignment introduced at the dynamical simulated annealing stage of the calculations. A total of 34 structures calculated by a hybrid distance geometry-dynamical simulated annealing method exhibit well-defined positions for both backbone and side-chain atoms. The average atomic root-mean-square difference between the individual structures and the minimized mean structure is 0.35(+/- 0.08) A for the backbone atoms and 0.89(+/- 0.17) A for all heavy atoms. The precision of the structure determination is discussed and correlated to the experimental input data.     

Comparison of the accuracy of protein solution structures derived from conventional and network-edited NOESY data.

Network-editing experiments are variants of the basic NOESY experiment that allow more accurate direct measurement of interproton distances in macromolecules by defeating specific spin-diffusion pathways. Two network-editing approaches, block-decoupled NOESY and complementary-block-decoupled-NOESY, were applied as three-dimensional, heteronuclear-edited experiments to distance measurement in a small protein, turkey ovomucoid third domain (OMTKY3). Two-hundred and twelve of the original 655 distance constraints observed in this molecule (Krezel AM et al., 1994, J Mol Biol 242:203-214) were improved by their replacement by distances derived from network-edited spectra, and distance geometry/simulated annealing solution structure calculations were performed from both the unimproved and improved distance sets. The resulting two families of structures were found to differ significantly, the most important differences being the hinge angle of a beta-turn and an expansion of the sampled conformation space in the region of the reactive-site loop. The structures calculated from network-editing data are interpreted as a more accurate model of the solution conformation of OMTKY3.     

Improved distance analysis in RNA using network-editing techniques for overcoming errors due to spin diffusion

Multispin magnetization transfer, or spin diffusion, is a significant source of error in NOESY-derived distance measurements for the determination of nucleic acid solution structures. The BD-NOESY and CBD-NOESY experiments, which allow the measurement of interproton distances with greatly reduced contributions from spin diffusion, have been adapted to structural analysis in RNA oligonucleotides. The techniques are applied to a lead-dependent ribozyme (LZ2). We demonstrate the measurement of both aromatic proton–aromatic proton NOEs free of spin diffusion involving the intervening ribose moieties and aromatic proton–ribose proton NOEs free of the efficient cross-relaxation within the ribose ring. In LZ2, the accuracy and precision of the resulting distances are significantly improved. We also find that, by allowing the use of longer mixing times with greater sensitivity, the experimental attenuation of spin diffusion in RNA increases the distance range of interactions that can be analyzed. This effect permits measurement of important long-range distances in LZ2 that are not accessible with standard techniques. Thus, these techniques allow the simultaneous optimization of the number, accuracy, and precision of distance constraints used for RNA structure determinations.     

Error distribution derived NOE distance restraints

Errors and imprecisions in distance restraints derived from NOESY peak volumes are usually accounted for by generous lower and upper bounds on the distances. In this paper, we propose a new form of distance restraints, replacing the subjective bounds by a potential function obtained from the error distribution of the distances. We derived the shape of the potential from molecular dynamics calculations and by comparison of NMR data with X-ray crystal structures. We used complete cross-validation to derive the optimal weight for the data in the calculation. In a model system with synthetic restraints, the accuracy of the structures improved significantly compared to calculations with the usual form of restraints. For experimental data sets, the structures systematically approach the X-ray crystal structures of the same protein. Also standard quality indicators improve compared to standard calculations. The results did not depend critically on the exact shape of the potential. The new approach is less subjective and uses fewer assumptions in the interpretation of NOESY peak volumes as distance restraints than the usual approach. Figures of merit for the structures, such as the RMS difference from the average structure or the RMS difference from the data, are therefore less biased and more meaningful measures of structure quality than with the usual form of restraints.     

NMR-pseudoenergy approach to the solution structure of acyl carrier protein

A method for protein structure determination from two-dimensional NMR cross-relaxation data is presented and explored by using short amino acid segments from acyl carrier protein as a test case. The method is based on a molecular mechanics program and incorporates NMR distance constraints in the form of a pseudoenergy term that accurately reflects the distance-dependent precision of NMR cross-relaxation data. When it is used in an indiscriminant fashion, the method has a tendency to produce structures representing local energy minima near starting structures, rather than structures representing a global energy minimum. However, stepwise inclusion of energy terms, beginning with a function heavily weighted by backbone distance constraints, appears to simplify the potential energy surface to a point where convergence to a common backbone structure from a variety of starting structures is possible. In the case of the segment from residues 3 to 15 in acyl carrier protein, a nearly perfect alpha-helix is produced starting with a linear chain, an alpha-helical chain, or a chain having residues with alternating linear and alpha-helical backbone torsional angles. In the case of the segment from residues 26 to 36 a structure having a right-handed loop is produced.     

Three-dimensional structure of a DNA hairpin in solution: two-dimensional NMR studies and distance geometry calculations on d(CGCGTTTTCGCG)

The three-dimensional structure of d(CGCGTTTTCGCG) in solution has been determined from proton NMR data by using distance geometry methods. The rate of dipolar cross-relaxation between protons close together in space is used to calculate distances between proton pairs within 5 A of each other; these distances are used as input to a distance geometry algorithm that embeds this distance matrix in three-dimensional space. The resulting refined structures that best agree with the input distances are all very similar to each other and show that the DNA sequence forms a hairpin in solution; the bases of the loop region are stacked, and the stem region forms a right-handed helix. The advantages and limitations of the technique, as well as the computer requirements of the algorithm, are discussed.     

Determination of the three-dimensional solution structure of ragweed allergen Amb t V by nuclear magnetic resonance spectroscopy.

Analysis of two-dimensional NMR experiments has afforded essentially complete assignment of all proton resonances in the allergenic protein Amb t V. Conformational constraints were obtained from the NMR data in three forms: interproton distances derived from NOE cross-peak intensities of NOESY spectra, torsion angle constraints derived from J-coupling constants of COSY and PE-COSY spectra, and hydrogen bond constraints derived from hydrogen-exchange experiments. Conformations of Amb t V with low constraint violations were generated using dynamic simulated annealing in the program XPLOR. The refined structures are comprised of a C-terminal alpha-helix, a short stretch of triple-stranded antiparallel beta-sheet, and several loops. In addition, the cystine partners of the four disulfide linkages (for which there are no biochemical data) have been assigned. The refined structures of Amb t V will allow us to suggest surface substructures for the Amb V allergens that are likely to participate in B cell epitopes and will assist us in defining the Ia/T cell epitopes that interact with the MHC class II (or Ia) molecule and the T cell receptor leading to the induction of the immune response to Amb t V.     

Solution conformation of salmon calcitonin in sodium dodecyl sulfate micelles as determined by two-dimensional NMR and distance geometry calculations

The 32 amino acid hormone salmon calcitonin was studied at pH 3.7 and 7.4 by two-dimensional NMR in sodium dodecyl sulfate (SDS) micelles at 310 K. The spectrum was fully assigned, and the secondary structure was obtained from nuclear Overhauser enhancement spectroscopy (NOESY), 3JHN alpha coupling constants, and slowly exchanging amide data. Three-dimensional structures consistent with NMR data were generated by using distance geometry calculations. A set of 260 interproton distances, derived from NOESY, and hydrogen-bond constraints, obtained from analysis of the amide exchange, were used. From the initial random conformations, 13 distance geometry structures with minimal violations were selected for further refinement with restrained energy minimization. In SDS, at both pHs, the main conformational feature of the hormone is an alpha-helix from Thr6 through Tyr22, thus including the amphipathic 8-22 segment and two residues of the Cys1-Cys7 N-terminal loop. The C-terminal decapeptide forms a loop folded back toward the helix. The biological significance of this conformation is discussed.     

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.     

Solution structures of proteins from NMR data and modeling: alternative folds for neutrophil peptide 5

The structure of neutrophil peptide 5 in solution has recently been reported (Pardi et al., 1988). The structure determination was accomplished by using a distance geometry algorithm and 107 interproton distance constraints obtained from 2D NMR data. In each of the eight independent solutions to the distance geometry equations, the overall fold of the polypeptide backbone was identical and the root mean square (rms) deviation between backbone atoms of the superimposed structures was small (approximately 2.4 A). In this paper we report additional NP-5 structures obtained by using a new structure generation algorithm: a Monte Carlo search in torsion angle space. These structures have a large rms backbone deviation from the distance geometry structures (approximately 5.0 A). The backbone topologies differ in significant respects from the distance geometry structures and from each other. Structures are found that are pseudo mirror images of part or all of the fold corresponding to that first obtained with the distance geometry procedure. For small proteins, the problem of distinguishing the correct structure among pseudo mirror images is likely to be greater than previously recognized. When a set of test distance constraints constructed from a novel Monte Carlo structure is used as input in the distance geometry algorithm, the fold of the resulting structure does not correspond to that of the target. The results also demonstrate that the previously accepted criteria (the magnitude of the rms deviation between multiple solutions of the distance geometry equations) for defining the accuracy and precision of a peptide structure generated from NMR data are inadequate. An energetic analysis of structures corresponding to the different folding topologies has been carried out. The molecular mechanics energies obtained by minimization and molecular dynamics refinement provide sufficient information to eliminate certain alternative structures. On the basis of a careful comparison of the different trial structures with the experimental data, it is concluded that the NP-5 peptide fold which was originally reported is most consistent with the data. An alternative fold corresponding to structures with low energies and small total distance violations is ruled out because for this fold predicted NOEs are not observed experimentally.     

Solution structure of human calcitonin in membrane-mimetic environment: The role of the amphipathic helix

The 32 amino acid hormone human calcitonin was studied at pH 3.7 and 7.4 by multidimensional NMR spectroscopy in sodium dodecyl sulfate micelles at 310K. The secondary structure was obtained from nuclear Overhauser enhancement spectroscopy (NOESY), ³J_HNα coupling constants, and slowly exchanging amide data. Three-dimensional structures consistent with NMR data were generated by using distance geometry calculations. A set of 265 interproton distances derived from NOESY experiments, hydrogen-bond constraints obtained from amide exchange, and coupling constants were used. From the initial random conformations, 30 distance geometry structures with minimal violations were selected for further refinement with restrained energy minimization. In micelles, at both pHs, the hormone assumes an amphipathic α-helix from Leu9 to Phe16, followed by a type-I β-turn between residues Phe16 and Phe19. From His20 onward the molecule is extended and no interaction with the helix was observed. The relevance of the amphipathic helix for the structure–activity relationship, the possible mechanisms of interaction with the receptor, as well as the formation of fibrillar aggregates, is discussed. Proteins 32:314–323, 1998. © 1998 Wiley-Liss, Inc.     

High resolution solution structure of apo calcyclin and structural variations in the S100 family of calcium-binding proteins

The three-dimensional solution structure of apo rabbit lung calcyclin has been refined to high resolution through the use of heteronuclear NMR spectroscopy and 13C,15N- enriched protein. Upon completing the assignment of virtually all of the 15N, 13C and 1H NMR resonances, the solution structure was determined from a combination of 2814 NOE- derived distance constraints, and 272 torsion angle constraints derived from scalar couplings. A large number of critical inter- subunit NOEs (386) were identified from 13C- select,13C-filtered NOESY experiments, providing a highly accurate dimer interface. The combination of distance geometry and restrained molecular dynamics calculations yielded structures with excellent agreement with the experimental data and high precision (rmsd from the mean for the backbone atoms in the eight helices: 0.33 Å). Calcyclin exhibits a symmetric dimeric fold of two identical 90 amino acid subunits, characteristic of the S100 subfamily of EF-hand Ca2+-binding proteins. The structure reveals a readily identified pair of putative sites for binding of Zn2+. In order to accurately determine the structural features that differentiate the various S100 proteins, distance difference matrices and contact maps were calculated for the NMR structural ensembles of apo calcyclin and rat and bovine S100B. These data show that the most significant variations among the structures are in the positioning of helix III and in loops, the regions with least sequence similarity. Inter-helical angles and distance differences for the proteins show that the positioning of helix III of calcyclin is most similar to that of bovine S100B, but that the helix interfaces are more closely packed in calcyclin than in either S100B structure. Surprisingly large differences were found in the positioning of helix III in the two S100B structures, despite there being only four non-identical residues, suggesting that one or both of the S100B structures requires further refinement.     

Determination of the complete three-dimensional structure of the alpha-amylase inhibitor tendamistat in aqueous solution by nuclear magnetic resonance and distance geometry

The complete three-dimensional structure of the alpha-amylase inhibitor Tendamistat in aqueous solution was determined by 1H nuclear magnetic resonance and distance geometry calculations using the program DISMAN. Compared to an earlier, preliminary determination of the polypeptide backbone conformation, stereo-specific assignments were obtained for 41 of the 89 prochiral groups in the protein, and a much more extensive set of experimental constraints was collected, including 842 distance constraints from nuclear Overhauser effects and over 100 supplementary constraints from spin-spin coupling constants and the identification of intramolecular hydrogen bonds. The complete protein molecule, including the amino acid side-chains is characterized by a group of nine structures corresponding to the results of the nine DISMAN calculations with minimal residual error functions. The average of the pairwise minimal root-mean-square distances among these nine structures is 0.85 A for the polypeptide backbone, and 1.52 A for all the heavy atoms. The procedures used for the structure determination are described and a detailed analysis is presented of correlations between the experimental input data and the precision of the structure determination.     

Refinement of the NMR solution structure of a protein to remove distortions arising from neglect of internal motion.

The effect of internal motion on the quality of a protein structure derived from nuclear magnetic resonance (NMR) cross relaxation has been investigated experimentally. Internal rotation of the tyrosine-31 ring of turkey ovomucoid third domain was found to mediate magnetization transfer; the effect led to underestimation of proton-proton distances in its immediate neighborhood. Experimental methods that distinguish pure cross relaxation from chemical exchange mediated cross relaxation were used to separate true distances from distorted ones. Uncorrected and corrected sets of distances, where the corrections took internal motion into account, each were used as input to a distance geometry program for structural modeling. Each set of distances yielded a family of similar (converged) structures. The two families of structures differed considerably (2 A) in the region of tyrosine-31. In addition, differences as large as 1 A were observed at other positions throughout the structure. These results emphasize the importance of analyzing the effects of internal motions in order to obtain more accurate NMR solution structures.     

NMR distance measurements in DNA duplexes: sugars and bases have the same correlation times

To evaluate whether the sugar moieties of short DNA duplexes exhibit local motion of sufficient amplitude to affect interproton distance measurements, we have carried out a series of time-dependent NOESY experiments at increasingly shorter mixing times on dodecamer DNA duplexes. By use of the cytosine H5-H6 vector as a known distance in the bases and the geminal 2'H-2'H vector as a known distance in the sugars, the corresponding apparent cross-relaxation rates were sampled at various mixing times. While the ratio of the inverse sixth power of these two fixed distances is in the range 6-7, when the system is sampled at 100 ms the apparent initial rate of growth of the 2'H-2'H NOESY crosspeak is only 1.9-2.0 times faster than that of the H5-H6 crosspeak--in agreement with the results of Clore and Gronenborn [Clore, G. M., & Gronenborn, A. M. (1984) FEBS Lett. 172, 219; (1984) FEBS Lett. 175, 117] and of Gronenborn and Clore [Gronenborn, A. M., & Clore, G. M. (1985) Prog. NMR Spectrosc. 17, 1]. This observation was interpreted to indicate the existence of internal mobility with a 3-fold shorter correlation time for the sugar moieties in DNA and led to the use of this shorter correlation time to estimate sugar-sugar proton distances and many sugar-base proton distances in subsequent DNA structure determination. We have examined 2'H-2"H cross-relaxation and H5-H6 cross-relaxation at 100, 90, 60, 30, and 15 ms in dodecamer DNA duplexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA

Combined automated NOE assignment and structure determination module (CANDID) is a new software for efficient NMR structure determination of proteins by automated assignment of the NOESY spectra. CANDID uses an iterative approach with multiple cycles of NOE cross-peak assignment and protein structure calculation using the fast DYANA torsion angle dynamics algorithm, so that the result from each CANDID cycle consists of exhaustive, possibly ambiguous NOE cross-peak assignments in all available spectra and a three-dimensional protein structure represented by a bundle of conformers. The input for the first CANDID cycle consists of the amino acid sequence, the chemical shift list from the sequence-specific resonance assignment, and listings of the cross-peak positions and volumes in one or several two, three or four-dimensional NOESY spectra. The input for the second and subsequent CANDID cycles contains the three-dimensional protein structure from the previous cycle, in addition to the complete input used for the first cycle. CANDID includes two new elements that make it robust with respect to the presence of artifacts in the input data, i.e. network-anchoring and constraint-combination, which have a key role in de novo protein structure determinations for the successful generation of the correct polypeptide fold by the first CANDID cycle. Network-anchoring makes use of the fact that any network of correct NOE cross-peak assignments forms a self-consistent set; the initial, chemical shift-based assignments for each individual NOE cross-peak are therefore weighted by the extent to which they can be embedded into the network formed by all other NOE cross-peak assignments. Constraint-combination reduces the deleterious impact of artifact NOE upper distance constraints in the input for a protein structure calculation by combining the assignments for two or several peaks into a single upper limit distance constraint, which lowers the probability that the presence of an artifact peak will influence the outcome of the structure calculation. CANDID test calculations were performed with NMR data sets of four proteins for which high-quality structures had previously been solved by interactive protocols, and they yielded comparable results to these reference structure determinations with regard to both the residual constraint violations, and the precision and accuracy of the atomic coordinates. The CANDID approach has further been validated by de novo NMR structure determinations of four additional proteins. The experience gained in these calculations shows that once nearly complete sequence-specific resonance assignments are available, the automated CANDID approach results in greatly enhanced efficiency of the NOESY spectral analysis. The fact that the correct fold is obtained in cycle 1 of a de novo structure calculation is the single most important advance achieved with CANDID, when compared with previously proposed automated NOESY assignment methods that do not use network-anchoring and constraint-combination.