BioMagResBank database with sets of experimental NMR constraints corresponding to the structures of over 1400 biomolecules deposited in the Protein Data Bank

Experimental constraints associated with NMR structures are available from the Protein Data Bank (PDB) in the form of `Magnetic Resonance' (MR) files. These files contain multiple types of data concatenated without boundary markers and are difficult to use for further research. Reported here are the results of a project initiated to annotate, archive, and disseminate these data to the research community from a searchable resource in a uniform format. The MR files from a set of 1410 NMR structures were analyzed and their original constituent data blocks annotated as to data type using a semi-automated protocol. A new software program called Wattos was then used to parse and archive the data in a relational database. From the total number of MR file blocks annotated as constraints, it proved possible to parse 84% (3337/3975). The constraint lists that were parsed correspond to three data types (2511 distance, 788 dihedral angle, and 38 residual dipolar couplings lists) from the three most popular software packages used in NMR structure determination: XPLOR/CNS (2520 lists), DISCOVER (412 lists), and DYANA/DIANA (405 lists). These constraints were then mapped to a developmental version of the BioMagResBank (BMRB) data model. A total of 31 data types originating from 16 programs have been classified, with the NOE distance constraint being the most commonly observed. The results serve as a model for the development of standards for NMR constraint deposition in computer-readable form. The constraints are updated regularly and are available from the BMRB web site (http://www.bmrb.wisc.edu).     

Protein loop closure using orientational restraints from NMR data

Protein loops often play important roles in biological functions. Modeling loops accurately is crucial to determining the functional specificity of a protein. Despite the recent progress in loop prediction approaches, which led to a number of algorithms over the past decade, few rigorous algorithmic approaches exist to model protein loops using global orientational restraints, such as those obtained from residual dipolar coupling (RDC) data in solution nuclear magnetic resonance (NMR) spectroscopy. In this article, we present a novel, sparse data, RDC‐based algorithm, which exploits the mathematical interplay between RDC‐derived sphero‐conics and protein kinematics, and formulates the loop structure determination problem as a system of low‐degree polynomial equations that can be solved exactly, in closed‐form. The polynomial roots, which encode the candidate conformations, are searched systematically, using provable pruning strategies that triage the vast majority of conformations, to enumerate or prune all possible loop conformations consistent with the data; therefore, completeness is ensured. Results on experimental RDC datasets for four proteins, including human ubiquitin, FF2, DinI, and GB3, demonstrate that our algorithm can compute loops with higher accuracy, a three‐ to six‐fold improvement in backbone RMSD, versus those obtained by traditional structure determination protocols on the same data. Excellent results were also obtained on synthetic RDC datasets for protein loops of length 4, 8, and 12 used in previous studies. These results suggest that our algorithm can be successfully applied to determine protein loop conformations, and hence, will be useful in high‐resolution protein backbone structure determination, including loops, from sparse NMR data. Proteins 2012. © 2011 Wiley Periodicals, Inc.     

BioMagResBank databases DOCR and FRED containing converted and filtered sets of experimental NMR restraints and coordinates from over 500 protein PDB structures

We present two new databases of NMR-derived distance and dihedral angle restraints: the Database Of Converted Restraints (DOCR) and the Filtered Restraints Database (FRED). These databases currently correspond to 545 proteins with NMR structures deposited in the Protein Databank (PDB). The criteria for inclusion were that these should be unique, monomeric proteins with author-provided experimental NMR data and coordinates available from the PDB capable of being parsed and prepared in a consistent manner. The Wattos program was used to parse the files, and the CcpNmr FormatConverter program was used to prepare them semi-automatically. New modules, including a new implementation of Aqua in the BioMagResBank (BMRB) software Wattos were used to analyze the sets of distance restraints (DRs) for inconsistencies, redundancies, NOE completeness, classification and violations with respect to the original coordinates. Restraints that could not be associated with a known nomenclature were flagged. The coordinates of hydrogen atoms were recalculated from the positions of heavy atoms to allow for a full restraint analysis. The DOCR database contains restraint and coordinate data that is made consistent with each other and with IUPAC conventions. The FRED database is based on the DOCR data but is filtered for use by test calculation protocols and longitudinal analyses and validations. These two databases are available from websites of the BMRB and the Macromolecular Structure Database (MSD) in various formats: NMR-STAR, CCPN XML, and in formats suitable for direct use in the software packages CNS and CYANA.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s10858-005-2195-0These authors contributed equally to this work.     

Towards structural genomics of RNA: rapid NMR resonance assignment and simultaneous RNA tertiary structure determination using residual dipolar couplings

We report a new residual dipolar couplings (RDCs) based NMR procedure for rapidly determining RNA tertiary structure demonstrated on a uniformly (15)N/(13)C-labeled 27 nt variant of the trans-activation response element (TAR) RNA from HIV-I. In this procedure, the time-consuming nuclear Overhauser enhancement (NOE)-based sequential assignment step is replaced by a fully automated RDC-based assignment strategy. This approach involves examination of all allowed sequence-specific resonance assignment permutations for best-fit agreement between measured RDCs and coordinates for sub-structures in a target RNA. Using idealized A-form geometries to model Watson-Crick helices and coordinates from a previous X-ray structure to model a hairpin loop in TAR, the best-fit RDC assignment solutions are determined very rapidly (相似文献   

A large data set comparison of protein structures determined by crystallography and NMR: statistical test for structural differences and the effect of crystal packing

The existence of a large number of proteins for which both nuclear magnetic resonance (NMR) and X-ray crystallographic coordinates have been deposited into the Protein Data Bank (PDB) makes the statistical comparison of the corresponding crystal and NMR structural models over a large data set possible, and facilitates the study of the effect of the crystal environment and other factors on structure. We present an approach for detecting statistically significant structural differences between crystal and NMR structural models which is based on structural superposition and the analysis of the distributions of atomic positions relative to a mean structure. We apply this to a set of 148 protein structure pairs (crystal vs NMR), and analyze the results in terms of methodological and physical sources of structural difference. For every one of the 148 structure pairs, the backbone root-mean-square distance (RMSD) over core atoms of the crystal structure to the mean NMR structure is larger than the average RMSD of the members of the NMR ensemble to the mean, with 76% of the structure pairs having an RMSD of the crystal structure to the mean more than a factor of two larger than the average RMSD of the NMR ensemble. On average, the backbone RMSD over core atoms of crystal structure to the mean NMR is approximately 1 A. If non-core atoms are included, this increases to 1.4 A due to the presence of variability in loops and similar regions of the protein. The observed structural differences are only weakly correlated with the age and quality of the structural model and differences in conditions under which the models were determined. We examine steric clashes when a putative crystalline lattice is constructed using a representative NMR structure, and find that repulsive crystal packing plays a minor role in the observed differences between crystal and NMR structures. The observed structural differences likely have a combination of physical and methodological causes. Stabilizing attractive interactions arising from intermolecular crystal contacts which shift the equilibrium of the crystal structure relative to the NMR structure is a likely physical source which can account for some of the observed differences. Methodological sources of apparent structural difference include insufficient sampling or other issues which could give rise to errors in the estimates of the precision and/or accuracy.     

Optimized set of two-dimensional experiments for fast sequential assignment,secondary structure determination,and backbone fold validation of 13C/15N-labelled proteins

NMR experiments are presented which allow backbone resonance assignment, secondary structure identification, and in favorable cases also molecular fold topology determination from a series of two-dimensional ¹H-¹⁵N HSQC-like spectra. The ¹H-¹⁵N correlation peaks are frequency shifted by an amount ± _X along the ¹⁵N dimension, where _X is the C, C, or H frequency of the same or the preceding residue. Because of the low dimensionality (2D) of the experiments, high-resolution spectra are obtained in a short overall experimental time. The whole series of seven experiments can be performed in typically less than one day. This approach significantly reduces experimental time when compared to the standard 3D-based methods. The here presented methodology is thus especially appealing in the context of high-throughput NMR studies of protein structure, dynamics or molecular interfaces.     

Filtering and selection of structural models: combining docking and NMR

It is generally accepted that protein structures are more conserved than protein sequences, and 3D structure determination by computer simulations have become an important necessity in the postgenomic area. Despite major successes no robust, fast, and automated ab initio prediction algorithms for deriving accurate folds of single polypeptide chains or structures of intermolecular complexes exist at present. Here we present a methodology that uses selection and filtering of structural models generated by docking of known substructures such as individual proteins or domains through easily obtainable experimental NMR constraints. In particular, residual dipolar couplings and chemical shift mapping are used. Heuristic inclusion of chemical or biochemical knowledge about point-to-point interactions is combined in our selection strategy with the NMR data and commonly used contact potentials. We demonstrate the approach for the determination of protein-protein complexes using the EIN/HPr complex as an example and for establishing the domain-domain orientation in a chimeric protein, the recently determined hybrid human-Escherichia. coli thioredoxin.     

Validating the use of database potentials in protein structure determination by NMR

The refinement of protein structures determined by nuclear magnetic resonance spectroscopy against database potentials of mean force allows for the exclusion of unfavourable conformations of the protein backbone during a structure calculation, resulting in protein structures with a marked improvement in Ramachandran statistics. In this communication, we use multiple sets of residual dipolar couplings as quality assessment criteria for several proteins and show that not only do the Ramachandran and structural quality statistics improve, but a significant improvement in the accuracy of structures is achieved upon refinement.     

NMR experiments for the measurement of proton-proton and carbon-carbon residual dipolar couplings in uniformly labelled oligosaccharides

A 2D-HSQC-carbon selective/proton selective-constant time COSY, 2D-HSQC-(sel C, sel H)-CT COSY experiment, which is applicable to uniformly ¹³C isotopically enriched samples (U-¹³C) of oligosaccharides or oligonucleotides is proposed for the measurement of proton–proton RDC in crowded regions of 2D-spectra. In addition, a heteronuclear constant time-COSY experiment, ¹³C-¹³C CT-COSY, is proposed for the measurement of one bond carbon–carbon RDC in these molecules. These two methods provide an extension, to U-¹³C molecules, of the original homonuclear constant time-COSY experiment proposed by Tian et al. (1999) for saccharides. The combination of a number of these RDC with NOE data may provide the method of choice to study oligosaccharide conformation in the free and receptor-bound state.     

A Thorough Dynamic Interpretation of Residual Dipolar Couplings in Ubiquitin

The presence of slow motions with large amplitudes, as detected by measurements based on residual dipolar couplings [Peti, W., Meiler, J., Brueschweiler, R. and Griesinger, C. (2002) J. Am. Chem. Soc., 124, 5822–5833], has stirred up much discussion in recent years. Based on ubiquitin NH residual dipolar couplings (rdcs) measured in 31 different alignment conditions, a model-free analysis of structure and dynamics [Meiler, J., Peti, W., Prompers, J., Griesinger, C. and Brueschweiler, R. (2001) J. Am. Chem. Soc., 123, 6098–6107] is presented. Starting from this broad experimental basis, rdc-based order parameters with so far unattained accuracy were determined. These rdc-based order parameters underpin the presence of new modes of motion slower than the inverse overall tumbling correlation time. Amplitudes and anisotropies of the motion were derived. The effect of structural noise on the results was proven to be negligible. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

SESAME: A least-squares approach to the evaluation of protein structures computed from NMR data

Summary A method is proposed for defining a probability distribution on an ensemble of protein conformations from a 2D NOE spectrum, while at the same time back-calculating the experimental spectrum from the ensemble. This enables one to assess the relative quality and significance of the conformations, and to test the consistency of the ensemble as a whole with the experimental spectrum. The method eliminates the need to integrate the cross-peak intensities and is surprisingly insensitive to random noise in the spectrum. In this communication, these advantages are demonstrated by applying the method to simulated data, for which the correct result is already known.Abbreviations NOE nuclear Overhauser effect - BPTI basic pancreatic trypsin inhibitor - rmsd root-mean-square deviation     

Structure determination of a new protein from backbone-centered NMR data and NMR-assisted structure prediction

Targeting of proteins for structure determination in structural genomic programs often includes the use of threading and fold recognition methods to exclude proteins belonging to well-populated fold families, but such methods can still fail to recognize preexisting folds. The authors illustrate here a method in which limited amounts of structural data are used to improve an initial homology search and the data are subsequently used to produce a structure by data-constrained refinement of an identified structural template. The data used are primarily NMR-based residual dipolar couplings, but they also include additional chemical shift and backbone-nuclear Overhauser effect data. Using this methodology, a backbone structure was efficiently produced for a 10 kDa protein (PF1455) from Pyrococcus furiosus. Its relationship to existing structures and its probable function are discussed.     

Various strategies of using residual dipolar couplings in NMR-driven protein docking: application to Lys48-linked di-ubiquitin and validation against 15N-relaxation data

When classical, Nuclear Overhauser Effect (NOE)-based approaches fail, it is possible, given high-resolution structures of the free molecules, to model the structure of a complex in solution based solely on chemical shift perturbation (CSP) data in combination with orientational restraints from residual dipolar couplings (RDCs) when available. RDCs can be incorporated into the docking following various strategies: as direct restraints and/or as intermolecular intervector projection angle restraints (Meiler et al., J Biomol NMR 2000;16:245-252). The advantage of the latter for docking is that they directly define the relative orientation of the molecules. A combined protocol in which RDCs are first introduced as intervector projection angle restraints and at a later stage as direct restraints is shown here to give the best performance. This approach, implemented in our information-driven docking approach HADDOCK (Dominguez et al., J Am Chem Soc 2003;125:1731-1737), is used to determine the solution structure of the Lys48-linked di-ubiquitin, for which chemical shift mapping, RDCs, and (15)N-relaxation data have been previously obtained (Varadan et al., J Mol Biol 2002;324:637-647). The resulting structures, derived from CSP and RDC data, are cross-validated using (15)N-relaxation data. The solution structure differs from the crystal structure by a 20 degrees rotation of the two ubiquitin units relative to each other.     

RCSB Protein Data bank: Tools for visualizing and understanding biological macromolecules in 3D

Now in its 52nd year of continuous operations, the Protein Data Bank (PDB) is the premiere open‐access global archive housing three‐dimensional (3D) biomolecular structure data. It is jointly managed by the Worldwide Protein Data Bank (wwPDB) partnership. The Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) is funded by the National Science Foundation, National Institutes of Health, and US Department of Energy and serves as the US data center for the wwPDB. RCSB PDB is also responsible for the security of PDB data in its role as wwPDB‐designated Archive Keeper. Every year, RCSB PDB serves tens of thousands of depositors of 3D macromolecular structure data (coming from macromolecular crystallography, nuclear magnetic resonance spectroscopy, electron microscopy, and micro‐electron diffraction). The RCSB PDB research‐focused web portal (RCSB.org) makes PDB data available at no charge and without usage restrictions to many millions of PDB data consumers around the world. The RCSB PDB training, outreach, and education web portal (PDB101.RCSB.org) serves nearly 700 K educators, students, and members of the public worldwide. This invited Tools Issue contribution describes how RCSB PDB (i) is organized; (ii) works with wwPDB partners to process new depositions; (iii) serves as the wwPDB‐designated Archive Keeper; (iv) enables exploration and 3D visualization of PDB data via RCSB.org; and (v) supports training, outreach, and education via PDB101.RCSB.org. New tools and features at RCSB.org are presented using examples drawn from high‐resolution structural studies of proteins relevant to treatment of human cancers by targeting immune checkpoints.     

NMR assignments, secondary structure, and global fold of calerythrin, an EF-hand calcium-binding protein from Saccharopolyspora erythraea

Calerythrin is a 20 kDa calcium-binding protein isolated from gram-positive bacterium Saccharopolyspora erythraea. Based on amino acid sequence homology, it has been suggested that calerythrin belongs to the family of invertebrate sarcoplasmic EF-hand calcium-binding proteins (SCPs), and therefore it is expected to function as a calcium buffer. NMR spectroscopy was used to obtain structural information on the protein in solution. Backbone and side chain 1H, 13C, and 15N assignments were obtained from triple resonance experiments HNCACB, HN(CO)CACB, HNCO, CC(CO)NH, and [15N]-edited TOCSY, and HCCH-TOCSY. Secondary structure was determined by using secondary chemical shifts and characteristic NOEs. In addition, backbone N-H residual dipolar couplings were measured from a spin-state selective [1H, 15N] correlation spectrum acquired from a sample dissolved in a dilute liquid crystal. Four EF-hand motifs with characteristic helix-loop-helix patterns were observed. Three of these are typical calcium-binding EF-hands, whereas site 2 is an atypical nonbinding site. The global fold of calerythrin was assessed by dipolar couplings. Measured dipolar couplings were compared with values calculated from four crystal structures of proteins with sequence homology to calerythrin. These data allowed us to recognize an overall similarity between the folds of calerythrin and sarcoplasmic calcium-binding proteins from the sandworm Nereis diversicolor and the amphioxus Branchiostoma lanceolatum.     

蛋白质溶液NMR结构测定的一些新进展

新的标记技术的进展和采用稀释的液晶作为溶剂以提供额外的结构信息,提高了核磁共振技术测定蛋白质溶液三维结构的精度,扩大了分子质量测定范围.目前已经利用多维 ¹⁵N,¹³C,²H标记NMR测定了许多分子质量为30 ku左右的蛋白质溶液结构,这一上限可能还会被进一步提高.     

High-resolution X-ray and NMR structures of the SMN Tudor domain: conformational variation in the binding site for symmetrically dimethylated arginine residues

The SMN protein, which is linked to spinal muscular atrophy (SMA), plays an important role in the assembly of the spliceosomal small nuclear ribonucleoprotein complexes. This function requires binding of SMN to the arginine-glycine (RG) rich C-terminal tails of the Sm proteins, which contain symmetrically dimethylated arginine residues (sDMA) in vivo. Using NMR titrations, we show that the SMN Tudor domain recognizes these sDMAs in the methylated RG repeats. Upon complex formation a cluster of conserved aromatic residues in the SMN Tudor domain interacts with the sDMA methyl groups. We present two high resolution structures of the uncomplexed SMN Tudor domain, a 1.8A crystal structure and an NMR structure that has been refined against a large number of backbone and side-chain residual dipolar couplings. The backbone conformation of both structures is very similar, however, differences are observed for the cluster of conserved aromatic side-chains in the sDMA binding pocket. In order to validate these variations we introduce a novel application of residual dipolar couplings for aromatic rings. We show that structural information can be derived from aromatic ring residual dipolar couplings, even in the presence of internal motions such as ring flipping. These residual dipolar couplings and ring current shifts independently confirm that the SMN Tudor domain adopts two different conformations in the sDMA binding pocket. The observed structural variations may play a role for the recognition of sDMAs.     

Electron paramagnetic resonance study of structural changes in the O photointermediate of bacteriorhodopsin

The structural changes of bacteriorhodopsin during its photochemical cycle, as revealed by crystal structures of trapped intermediates, have provided insights to the proton translocation mechanism. Because accumulation of the last photointermediate, O, appears to be hindered by lattice forces in the crystals, the only information about the structure of this state is from suggested analogies with the determined structures of the non-illuminated D85S mutant and wild-type bacteriorhodopsin at low pH. We used electron paramagnetic resonance spectroscopy of site-directed spin labels at the extracellular protein surface in membranes to test these models. Spin-spin dipolar interactions in the authentic O state compared to the non-illuminated state revealed that the distance between helices C and F increases by ca 4 Angstroms, there is no distance change between helices D and F, and the distance between helix D and helix B of the adjacent monomer increases. Further, the mobility changes of single labels indicate that helices E and F move outward from the proton channel at the center of the protein, and helix D tilts inward. The overall pattern of movements suggests that the model at acid pH is a better representation of the O state than D85S. However, the mobility analysis of spin-labels on the B-C interhelical loop indicates that the antiparallel beta-sheet maintains its ordered secondary structure in O, instead of the predicted disorder in the two structural models. During decay of the O state, the last step of the photocycle, a proton is transferred from Asp85 to proton release complex in the extracellular proton channel. The structural changes in O suggest the need of large conformational changes to drive the Arg82 side-chain back to its initial orientation towards Asp85, and to rearrange the numerous water molecules in this region in order to conduct the proton away from Asp85.     

DRESS: a database of REfined solution NMR structures

Several studies have shown that biomolecular NMR structures are often of lower quality when compared to crystal structures, and consequently they are often excluded from structural analyses. We present a publicly available database of re-refined NMR structures, exhibiting significantly improved quality. This database (available at http://www.cmbi.kun.nl/dress/) presents a uniformly refined and validated set of structural models that improves the value of these NMR structures as input for experimental and theoretical studies in many fields of research.     

NMR studies of succinoglycan repeating-unit octasaccharides from Rhizobium meliloti and Agrobacterium radiobacter

Complete ¹H and ¹³C-nuclear magnetic resonance assignments have been obtained for the octasaccharide repeating units of the bacterial polysaccharide succinoglycan from Rhizobium meliloti Rm1021 and Agrobacterium radiobacter NCIB 11883. The assignments were used to determine the locations of the O-succinyl and O-acetyl substituents. The O-acetyl substituent in Rm1021 was attached to the 3rd residue from the reducing end, and the O-succinyl group was attached to the 7th residue in both octasaccharides. The structure of the Rm1021 octasaccharide is as shown below:

A small amount of succinate was also attached to C6 of the 6th residue in both octasaccharides.