A graphical method for analyzing distance restraints using residual dipolar couplings for structure determination of symmetric protein homo-oligomers

High-resolution structure determination of homo-oligomeric protein complexes remains a daunting task for NMR spectroscopists. Although isotope-filtered experiments allow separation of intermolecular NOEs from intramolecular NOEs and determination of the structure of each subunit within the oligomeric state, degenerate chemical shifts of equivalent nuclei from different subunits make it difficult to assign intermolecular NOEs to nuclei from specific pairs of subunits with certainty, hindering structural analysis of the oligomeric state. Here, we introduce a graphical method, DISCO, for the analysis of intermolecular distance restraints and structure determination of symmetric homo-oligomers using residual dipolar couplings. Based on knowledge that the symmetry axis of an oligomeric complex must be parallel to an eigenvector of the alignment tensor of residual dipolar couplings, we can represent distance restraints as annuli in a plane encoding the parameters of the symmetry axis. Oligomeric protein structures with the best restraint satisfaction correspond to regions of this plane with the greatest number of overlapping annuli. This graphical analysis yields a technique to characterize the complete set of oligomeric structures satisfying the distance restraints and to quantitatively evaluate the contribution of each distance restraint. We demonstrate our method for the trimeric E. coli diacylglycerol kinase, addressing the challenges in obtaining subunit assignments for distance restraints. We also demonstrate our method on a dimeric mutant of the immunoglobulin-binding domain B1 of streptococcal protein G to show the resilience of our method to ambiguous atom assignments. In both studies, DISCO computed oligomer structures with high accuracy despite using ambiguously assigned distance restraints.     

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

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

Structure determination of symmetric homo-oligomers by a complete search of symmetry configuration space, using NMR restraints and van der Waals packing

Structural studies of symmetric homo-oligomers provide mechanistic insights into their roles in essential biological processes, including cell signaling and cellular regulation. This paper presents a novel algorithm for homo-oligomeric structure determination, given the subunit structure, that is both complete, in that it evaluates all possible conformations, and data-driven, in that it evaluates conformations separately for consistency with experimental data and for quality of packing. Completeness ensures that the algorithm does not miss the native conformation, and being data-driven enables it to assess the structural precision possible from data alone. Our algorithm performs a branch-and-bound search in the symmetry configuration space, the space of symmetry axis parameters (positions and orientations) defining all possible C(n) homo-oligomeric complexes for a given subunit structure. It eliminates those symmetry axes inconsistent with intersubunit nuclear Overhauser effect (NOE) distance restraints and then identifies conformations representing any consistent, well-packed structure to within a user-defined similarity level. For the human phospholamban pentamer in dodecylphosphocholine micelles, using the structure of one subunit determined from a subset of the experimental NMR data, our algorithm identifies a diverse set of complex structures consistent with the nine intersubunit NOE restraints. The distribution of determined structures provides an objective characterization of structural uncertainty: backbone RMSD to the previously determined structure ranges from 1.07 to 8.85 A, and variance in backbone atomic coordinates is an average of 12.32 A(2). Incorporating vdW packing reduces structural diversity to a maximum backbone RMSD of 6.24 A and an average backbone variance of 6.80 A(2). By comparing data consistency and packing quality under different assumptions of oligomeric number, our algorithm identifies the pentamer as the most likely oligomeric state of phospholamban, demonstrating that it is possible to determine the oligomeric number directly from NMR data. Additional tests on a number of homo-oligomers, from dimer to heptamer, similarly demonstrate the power of our method to provide unbiased determination and evaluation of homo-oligomeric complex structures.     

RDC-assisted modeling of symmetric protein homo-oligomers

Protein oligomerization serves an important function in biological processes, yet solving structures of protein oligomers has always been a challenge. For solution NMR, the challenge arises both from the increased size of these systems and, in the case of homo-oligomers, from ambiguities in assignment of intra- as opposed to intersubunit NOEs. In this study, we present a residual dipolar coupling (RDC)-assisted method for constructing models of homo-oligomers with purely rotational symmetry. Utilizing the fact that one of the principal axes of the tensor describing the alignment needed for RDC measurement is always parallel to the oligomer symmetry axis, it is possible to greatly restrict possible models for the oligomer. Here, it is shown that, if the monomer structure is known, all allowed dimer models can be constructed using a grid search algorithm and evaluated based on RDC simulations and the quality of the interface between the subunits. Using the Bacillus subtilis protein YkuJ as an example, it is shown that the evaluation criteria based on just two sets of NH RDCs are very selective and can unambiguously produce a model in good agreement with an existing X-ray structure of YkuJ.     

Three-dimensional structure of the weakly associated protein homodimer SeR13 using RDCs and paramagnetic surface mapping

The traditional NMR‐based method for determining oligomeric protein structure usually involves distinguishing and assigning intra‐ and intersubunit NOEs. This task becomes challenging when determining symmetric homo‐dimer structures because NOE cross‐peaks from a given pair of protons occur at the same position whether intra‐ or intersubunit in origin. While there are isotope‐filtering strategies for distinguishing intra from intermolecular NOE interactions in these cases, they are laborious and often prove ineffectual in cases of weak dimers, where observation of intermolecular NOEs is rare. Here, we present an efficient procedure for weak dimer structure determination based on residual dipolar couplings (RDCs), chemical shift changes upon dilution, and paramagnetic surface perturbations. This procedure is applied to the Northeast Structural Genomics Consortium protein target, SeR13, a negatively charged Staphylococcus epidermidis dimeric protein (K_d 3.4 ± 1.4 mM) composed of 86 amino acids. A structure determination for the monomeric form using traditional NMR methods is presented, followed by a dimer structure determination using docking under orientation constraints from RDCs data, and scoring under residue pair potentials and shape‐based predictions of RDCs. Validation using paramagnetic surface perturbation and chemical shift perturbation data acquired on sample dilution is also presented. The general utility of the dimer structure determination procedure and the possible relevance of SeR13 dimer formation are discussed.     

Exact Solutions for Internuclear Vectors and Backbone Dihedral Angles from NH Residual Dipolar Couplings in Two Media, and their Application in a Systematic Search Algorithm for Determining Protein Backbone Structure

We have derived a quartic equation for computing the direction of an internuclear vector from residual dipolar couplings (RDCs) measured in two aligning media, and two simple trigonometric equations for computing the backbone (phi,psi) angles from two backbone vectors in consecutive peptide planes. These equations make it possible to compute, exactly and in constant time, the backbone (phi,psi) angles for a residue from RDCs in two media on any single backbone vector type. Building upon these exact solutions we have designed a novel algorithm for determining a protein backbone substructure consisting of alpha-helices and beta-sheets. Our algorithm employs a systematic search technique to refine the conformation of both alpha-helices and beta-sheets and to determine their orientations using exclusively the angular restraints from RDCs. The algorithm computes the backbone substructure employing very sparse distance restraints between pairs of alpha-helices and beta-sheets refined by the systematic search. The algorithm has been demonstrated on the protein human ubiquitin using only backbone NH RDCs, plus twelve hydrogen bonds and four NOE distance restraints. Further, our results show that both the global orientations and the conformations of alpha-helices and beta-strands can be determined with high accuracy using only two RDCs per residue. The algorithm requires, as its input, backbone resonance assignments, the identification of alpha-helices and beta-sheets as well as sparse NOE distance and hydrogen bond restraints.     

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.     

Protein side-chain resonance assignment and NOE assignment using RDC-defined backbones without TOCSY data

One bottleneck in NMR structure determination lies in the laborious and time-consuming process of side-chain resonance and NOE assignments. Compared to the well-studied backbone resonance assignment problem, automated side-chain resonance and NOE assignments are relatively less explored. Most NOE assignment algorithms require nearly complete side-chain resonance assignments from a series of through-bond experiments such as HCCH-TOCSY or HCCCONH. Unfortunately, these TOCSY experiments perform poorly on large proteins. To overcome this deficiency, we present a novel algorithm, called Nasca (NOE Assignment and Side-Chain Assignment), to automate both side-chain resonance and NOE assignments and to perform high-resolution protein structure determination in the absence of any explicit through-bond experiment to facilitate side-chain resonance assignment, such as HCCH-TOCSY. After casting the assignment problem into a Markov Random Field (MRF), Nasca extends and applies combinatorial protein design algorithms to compute optimal assignments that best interpret the NMR data. The MRF captures the contact map information of the protein derived from NOESY spectra, exploits the backbone structural information determined by RDCs, and considers all possible side-chain rotamers. The complexity of the combinatorial search is reduced by using a dead-end elimination (DEE) algorithm, which prunes side-chain resonance assignments that are provably not part of the optimal solution. Then an A* search algorithm is employed to find a set of optimal side-chain resonance assignments that best fit the NMR data. These side-chain resonance assignments are then used to resolve the NOE assignment ambiguity and compute high-resolution protein structures. Tests on five proteins show that Nasca assigns resonances for more than 90% of side-chain protons, and achieves about 80% correct assignments. The final structures computed using the NOE distance restraints assigned by Nasca have backbone RMSD 0.8–1.5 Å from the reference structures determined by traditional NMR approaches.     

NMR structure of cysteinyl-phosphorylated enzyme IIB of the N,N'-diacetylchitobiose-specific phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli

The determination by NMR of the solution structure of the phosphorylated enzyme IIB (P-IIB(Chb)) of the N,N'-diacetylchitobiose-specific phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli is presented. Most of the backbone and side-chain resonances were assigned using a variety of mostly heteronuclear NMR experiments. The remaining resonances were assigned with the help of the structure calculations.NOE-derived distance restraints were used in distance geometry calculations followed by molecular dynamics and simulated annealing protocols. In addition, combinations of ambiguous restraints were used to resolve ambiguities in the NOE assignments. By combining sets of ambiguous and unambiguous restraints into new ambiguous restraints, an error function was constructed that was less sensitive to information loss caused by assignment uncertainties. The final set of structures had a pairwise rmsd of 0.59 A and 1.16 A for the heavy atoms of the backbone and side-chains, respectively.Comparing the P-IIB(Chb) solution structure with the previously determined NMR and X-ray structures of the wild-type and the Cys10Ser mutant shows that significant differences between the structures are limited to the active-site region. The phosphoryl group at the active-site cysteine residue is surrounded by a loop formed by residues 10 through 16. NOE and chemical shift data suggest that the phosphoryl group makes hydrogen bonds with the backbone amide protons of residues 12 and 15. The binding mode of the phosphoryl group is very similar to that of the protein tyrosine phosphatases. The differences observed are in accordance with the presumption that IIB(Chb) has to be more resistant to hydrolysis than the protein tyrosine phosphatases. We propose a proton relay network by which a transfer occurs between the cysteine SH proton and the solvent via the hydroxyl group of Thr16.     

3d haro-Noesy-ch3nh and caro-Noesy-ch3nh Experiments for Double Labeled Proteins

Precision in the determination of the 3D structures of proteins by NMR depends on obtaining an adequate number of NOE restraints. Ambiguity in the assignment of NOE cross peaks between aromatic and other protons is an impediment to high quality structure determination. Two pulse sequences, 3D H_aro-NOESY-CH₃NH and 3D C_aro-NOESY-CH₃NH, based on a modification of a technique for simultaneous detection of ¹³C-¹H (of CH₃) and ¹⁵N-¹H correlations in one measurement, are proposed in the present work. These 3D experiments, which are optimized for resolution in the ¹³C and ¹⁵N dimensions, provide NOE information between aromatic protons and methyl or amide protons. CH₂ moieties are filtered out and the CH groups in aromatic rings are selected, allowing their NOE cross peaks to be unambiguously assigned. Unambiguous NOEs connecting aromatic and methyl or amide protons will provide important restraints for protein structure calculations.     

Time- and ensemble-averaged direct NOE restraints

Summary NMR data are collected as time- and ensemble-averaged quantities. Yet, in commonly used methods for structure determination of biomolecules, structures are required to satisfy simultaneously a large number of constrainsts. Recently, however, methods have been developed that allow a better fit of the experimental data by the use of time- or ensemble-averaged restraints. Thus far, these methods have been applied to structure refinement using distance and J-coupling restraints. In this paper, time and ensemble averaging is extended to the direct refinement with experimental NOE data. The implementation of time- and ensemble-averaged NOE restraints in DINOSAUR is described and illustrated with experimental NMR data for crambin, a 46-residue protein. Structure refinement with both time- and ensemble-averaged NOE restraints results in lower R-factors, indicating a better fit of the experimental NOE data.     

Structural model of the transmembrane Fo rotary sector of H+-transporting ATP synthase derived by solution NMR and intersubunit cross-linking in situ

H(+)-transporting, F(1)F(o)-type ATP synthases utilize a transmembrane H(+) potential to drive ATP formation by a rotary catalytic mechanism. ATP is formed in alternating beta subunits of the extramembranous F(1) sector of the enzyme, synthesis being driven by rotation of the gamma subunit in the center of the F(1) molecule between the alternating catalytic sites. The H(+) electrochemical potential is thought to drive gamma subunit rotation by first coupling H(+) transport to rotation of an oligomeric rotor of c subunits within the transmembrane F(o) sector. The gamma subunit is forced to turn with the c-oligomeric rotor due to connections between subunit c and the gamma and epsilon subunits of F(1). In this essay we will review recent studies on the Escherichia coli F(o) sector. The monomeric structure of subunit c, determined by NMR, shows that subunit c folds in a helical hairpin with the proton carrying Asp(61) centered in the second transmembrane helix (TMH). A model for the structural organization of the c(10) oligomer in F(o) was deduced from extensive cross-linking studies and by molecular modeling. The model indicates that the H(+)-carrying carboxyl of subunit c is occluded between neighboring subunits of the c(10) oligomer and that two c subunits pack in a "front-to-back" manner to form the H(+) (cation) binding site. In order for protons to gain access to Asp(61) during the protonation/deprotonation cycle, we propose that the outer, Asp(61)-bearing TMH-2s of the c-ring and TMHs from subunits composing the inlet and outlet channels must turn relative to each other, and that the swiveling motion associated with Asp(61) protonation/deprotonation drives the rotation of the c-ring. The NMR structures of wild-type subunit c differs according to the protonation state of Asp(61). The idea that the conformational state of subunit c changes during the catalytic cycle is supported by the cross-linking evidence in situ, and two recent NMR structures of functional mutant proteins in which critical residues have been switched between TMH-1 and TMH-2. The structural information is considered in the context of the possible mechanism of rotary movement of the c(10) oligomer during coupled synthesis of ATP.     

One- and two-dimensional 1H-NMR investigations of two 19-base-pair analogues of the tet operator

Two 19-base-pair oligodeoxynucleotides, analogues of one of the operators which specifically bind the repressor protein in the regulatory part of the transposon Tn10 tetracycline-resistance (tet) determinant, have been studied by 1H-NMR spectroscopy. The analogues contain a mismatch in the central base pair of the double helix (T.T or A.A). The imino protons have been assigned to the base pairs by one-dimensional NOE measurements, and the thermally induced transition from the duplex to the single strand has been followed. The cytidine amino resonances have been assigned by means of two-dimensional NOE spectroscopy in H2O. Two-dimensional phase-sensitive NOE and magnitude-correlated spectra have been recorded in 2H2O; all nonexchangeable protons, with the exception of some of H5', H5" protons, have been assigned. The NMR data made it possible to carry out a qualitative analysis of the structures of both oligodeoxynucleotides. The general structures close to B-DNA, show irregularities in the mismatch areas.     

NMR structure determination of a membrane protein with two transmembrane helices in micelles: MerF of the bacterial mercury detoxification system

The three-dimensional backbone structure of a membrane protein with two transmembrane helices in micelles was determined using solution NMR methods that rely on the measurement of backbone (1)H-(15)N residual dipolar couplings (RDCs) from samples of two different constructs that align differently in stressed polyacrylamide gels. Dipolar wave fitting to the (1)H-(15)N RDCs determines the helical boundaries based on periodicity and was utilized in the generation of supplemental dihedral restraints for the helical segments. The (1)H-(15)N RDCs and supplemental dihedral restraints enable the determination of the structure of the helix-loop-helix core domain of the mercury transport membrane protein MerF with a backbone RMSD of 0.58 A. Moreover, the fold of this polypeptide demonstrates that the two vicinal pairs of cysteine residues, shown to be involved in the transport of Hg(II) across the membrane, are exposed to the cytoplasm. This finding differs from earlier structural and mechanistic models that were based primarily on the somewhat atypical hydropathy plot for MerF and related transport proteins.     

A calculation strategy for the structure determination of symmetric demers by 1H NMR

The structure determination of symmetric dimers by NMR is impeded by the ambiguity of inter- and intramonomer NOE crosspeaks. In this paper, a calculation strategy is presented that allows the calculation of dimer structures without resolving ther ambuguity by additional experiments (like asymmetric labeling). The strategy employs a molecular dynamic-based simulated annealing approach to minimize a traget function. The experimental part of the target function contains distance restraints that correctly describe the ambiguity of the NOE peaks, and a novel term that restrains the symmetry of the dimer without requiring the knowledge of the symmetry axis. The use of the method is illustrated by three examples, using experimentally obtained data and model data derived from a known structure. For the purpose of testing the method, it is assumed that every NOE crosspeak is ambiguous in all three cases. It is shown that the structure of a homologous protein is known and in ab intio structure determination. The method can be extended to higher order symmetric multimers. © 1993 Wiley-Liss, Inc.     

Determination of solution structure and lipid micelle location of an engineered membrane peptide by using one NMR experiment and one sample

Antimicrobial peptides are universal host defense membrane-targeting molecules in a variety of life forms. Structure elucidation provides important insight into the mechanism of action. Here we present the three-dimensional structure of a membrane peptide in complex with dioctanoyl phosphatidylglycerol (D8PG) micelles determined by solution NMR spectroscopy. The model peptide, derived from the key antibacterial region of human LL-37, adopted an amphipathic helical structure based on 182 NOE-generated distance restraints and 34 chemical shift-derived angle restraints. Using the same NOESY experiment, it is also possible to delineate in detail the location of this peptide in lipid micelles via one-dimensional slice analysis of the intermolecular NOE cross peaks between the peptide and lipid. Hydrophobic aromatic side chains gave medium to strong NOE cross peaks, backbone amide protons and interfacial arginine side chain HN protons showed weak cross peaks, and arginine side chains on the hydrophilic face yielded no cross peaks with D8PG. Such a peptide-lipid intermolecular NOE pattern indicates a surface location of the amphipathic helix on the lipid micelle. In contrast, the epsilon HN protons of the three arginine side chains showed more or less similar intermolecular NOE cross peaks with lipid acyl chains when the helical structure was disrupted by selective d-amino acid incorporation, providing the basis for the selective toxic effect of the peptide against bacteria but not human cells. The differences in the intermolecular NOE patterns indicate that these peptides interact with model membranes in different mechanisms. Major NMR experiments for detecting protein-lipid NOE cross peaks are discussed.     

Refining the overall structure and subdomain orientation of ribosomal protein S4 delta41 with dipolar couplings measured by NMR in uniaxial liquid crystalline phases.

Prokaryotic protein S4 initiates assembly of the small ribosomal subunit by binding to 16 S rRNA. Residues 43-200 of S4 from Bacillus stearothermophilus (S4 Delta41) bind to both 16 S rRNA and to a mRNA pseudoknot. In order to obtain structure-based insights regarding RNA binding, we previously determined the solution structure of S4 Delta41 using NOE, hydrogen bond, and torsion angle restraints. S4 Delta41 is elongated, with two distinct subdomains, one all helical, the other including a beta-sheet. In contrast to the high resolution structures obtained for each individual subdomain, their relative orientation was not precisely defined because only 17 intersubdomain NOE restraints were determined. Compared to the 1.7 A crystal structure, when the sheet-containing subdomains are superimposed, the helical subdomain is twisted by almost 45 degrees about the long axis of the molecule in the solution structure. Because variations in subdomain orientation may explain how the protein recognizes multiple RNA targets, our current goal is to determine the orientation of the subdomains in solution with high precision. To this end, NOE assignments were re-examined. NOESY experiments on a specifically labeled sample revealed that one of the intersubdomain restraints had been misassigned. However, the revised set of NOE restraints produces solution structures that still have imprecisely defined subdomain orientations and that lie between the original NMR structure and the crystal structure. In contrast, augmenting the NOE restraints with N-H dipolar couplings, measured in uniaxial liquid crystalline phases, clearly establishes the relative orientation of the subdomains. Data obtained from two independent liquid crystalline milieux, DMPC/DHPC bicelles and the filamentous bacteriophage Pf1, show that the relative orientation of the subdomains in solution is quite similar to the subdomain orientation in the crystal structure. The solution structure, refined with dipolar data, is presented and its implications for S4's RNA binding activity are discussed.     

Determination of solution structure and lipid micelle location of an engineered membrane peptide by using one NMR experiment and one sample

Antimicrobial peptides are universal host defense membrane-targeting molecules in a variety of life forms. Structure elucidation provides important insight into the mechanism of action. Here we present the three-dimensional structure of a membrane peptide in complex with dioctanoyl phosphatidylglycerol (D8PG) micelles determined by solution NMR spectroscopy. The model peptide, derived from the key antibacterial region of human LL-37, adopted an amphipathic helical structure based on 182 NOE-generated distance restraints and 34 chemical shift-derived angle restraints. Using the same NOESY experiment, it is also possible to delineate in detail the location of this peptide in lipid micelles via one-dimensional slice analysis of the intermolecular NOE cross peaks between the peptide and lipid. Hydrophobic aromatic side chains gave medium to strong NOE cross peaks, backbone amide protons and interfacial arginine side chain H^N protons showed weak cross peaks, and arginine side chains on the hydrophilic face yielded no cross peaks with D8PG. Such a peptide-lipid intermolecular NOE pattern indicates a surface location of the amphipathic helix on the lipid micelle. In contrast, the εH^N protons of the three arginine side chains showed more or less similar intermolecular NOE cross peaks with lipid acyl chains when the helical structure was disrupted by selective d-amino acid incorporation, providing the basis for the selective toxic effect of the peptide against bacteria but not human cells. The differences in the intermolecular NOE patterns indicate that these peptides interact with model membranes in different mechanisms. Major NMR experiments for detecting protein-lipid NOE cross peaks are discussed.     

RECOORD: a recalculated coordinate database of 500+ proteins from the PDB using restraints from the BioMagResBank

State-of-the-art methods based on CNS and CYANA were used to recalculate the nuclear magnetic resonance (NMR) solution structures of 500+ proteins for which coordinates and NMR restraints are available from the Protein Data Bank. Curated restraints were obtained from the BioMagResBank FRED database. Although the original NMR structures were determined by various methods, they all were recalculated by CNS and CYANA and refined subsequently by restrained molecular dynamics (CNS) in a hydrated environment. We present an extensive analysis of the results, in terms of various quality indicators generated by PROCHECK and WHAT_CHECK. On average, the quality indicators for packing and Ramachandran appearance moved one standard deviation closer to the mean of the reference database. The structural quality of the recalculated structures is discussed in relation to various parameters, including number of restraints per residue, NOE completeness and positional root mean square deviation (RMSD). Correlations between pairs of these quality indicators were generally low; for example, there is a weak correlation between the number of restraints per residue and the Ramachandran appearance according to WHAT_CHECK (r = 0.31). The set of recalculated coordinates constitutes a unified database of protein structures in which potential user- and software-dependent biases have been kept as small as possible. The database can be used by the structural biology community for further development of calculation protocols, validation tools, structure-based statistical approaches and modeling. The RECOORD database of recalculated structures is publicly available from http://www.ebi.ac.uk/msd/recoord.     

Determination of the three-dimensional solution structure of the C-terminal domain of cellobiohydrolase I from Trichoderma reesei. A study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing

The solution structure of a synthetic 36-residue polypeptide comprising the C-terminal cellulose binding domain of cellobiohydrolase I (CT-CBH I) from Trichoderma reesei was investigated by nuclear magnetic resonance (NMR) spectroscopy. The 1H NMR spectrum was completely assigned in a sequential manner by two-dimensional NMR techniques. A large number of stereospecific assignments for beta-methylene protons, as well as ranges for the phi, psi, and chi 1 torsion angles, were obtained on the basis of sequential and intraresidue nuclear Overhauser enhancement (NOE) and coupling constant data in combination with a conformational data base search. The structure calculations were carried out in an iterative manner by using the hybrid distance geometry-dynamical simulated annealing method. This involved computing a series of initial structures from a subset of the experimental data in order to resolve ambiguities in the assignments of some NOE cross-peaks arising from chemical shift degeneracy. Additionally, this permitted us to extend the stereospecific assignments to the alpha-methylene protons of glycine using information on phi torsion angles derived from the initial structure calculations. The final experimental data set consisted of 554 interproton distance restraints, 24 restraints for 12 hydrogen bonds, and 33 phi, 24 psi, and 25 chi 1 torsion angle restraints. CT-CBH I has two disulfide bridges whose pairing was previously unknown. Analysis of structures calculated with all three possible combinations of disulfide bonds, as well as without disulfide bonds, indicated that the correct disulfide bridge pairing was 8-25 and 19-35. Forty-one structures were computed with the 8-25 and 19-35 disulfide bridges, and the average atomic rms difference between the individual structures and the mean structure obtained by averaging their coordinates was 0.33 +/- 0.04 A for the backbone atoms and 0.52 +/- 0.06 A for all atoms. The protein has a wedgelike shape with an amphiphilic character, one face being predominantly hydrophilic and the other mainly hydrophobic. The principal element of secondary structure is made up of an irregular triple-stranded antiparallel beta-sheet composed of residues 5-9 (beta 1), 24-28 (beta 2), and 33-36 (beta 3) in which strand beta 3 is hydrogen bonded to the other two strands.(ABSTRACT TRUNCATED AT 400 WORDS)