Frank Delaglio

NMRPipe: A multidimensional spectral processing system based on UNIX pipes

SummaryThe NMRPipe system is a UNIX software environment of processing, graphics, and analysis tools designed to meet current routine and research-oriented multidimensional processing requirements, and to anticipate and accommodate future demands and developments. The system is based on UNIX pipes, which allow programs running simultaneously to exchange streams of data under user control. In an NMRPipe processing scheme, a stream of spectral data flows through a pipeline of processing programs, each of which performs one component of the overall scheme, such as Fourier transformation or linear prediction. Complete multidimensional processing schemes are constructed as simple UNIX shell scripts. The processing modules themselves maintain and exploit accurate records of data sizes, detection modes, and calibration information in all dimensions, so that schemes can be constructed without the need to explicitly define or anticipate data sizes or storage details of real and imaginary channels during processing. The asynchronous pipeline scheme provides other substantial advantages, including high flexibility, favorable processing speeds, choice of both all-in-memory and disk-bound processing, easy adaptation to different data formats, simpler software development and maintenance, and the ability to distribute processing tasks on multi-CPU computers and computer networks.

Protein backbone angle restraints from searching a database for chemical shift and sequence homology

Chemical shifts of backbone atoms in proteins are exquisitely sensitive to local conformation, and homologous proteins show quite similar patterns of secondary chemical shifts. The inverse of this relation is used to search a database for triplets of adjacent residues with secondary chemical shifts and sequence similarity which provide the best match to the query triplet of interest. The database contains 13Cα, 13Cβ, 13C′, 1Hα and 15N chemical shifts for 20 proteins for which a high resolution X-ray structure is available. The computer program TALOS was developed to search this database for strings of residues with chemical shift and residue type homology. The relative importance of the weighting factors attached to the secondary chemical shifts of the five types of resonances relative to that of sequence similarity was optimized empirically. TALOS yields the 10 triplets which have the closest similarity in secondary chemical shift and amino acid sequence to those of the query sequence. If the central residues in these 10 triplets exhibit similar φ and Ψ backbone angles, their averages can reliably be used as angular restraints for the protein whose structure is being studied. Tests carried out for proteins of known structure indicate that the root-mean-square difference (rmsd) between the output of TALOS and the X-ray derived backbone angles is about 15°. Approximately 3% of the predictions made by TALOS are found to be in error.

TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts

NMR chemical shifts in proteins depend strongly on local structure. The program TALOS establishes an empirical relation between 13C, 15N and 1H chemical shifts and backbone torsion angles ϕ and ψ (Cornilescu et al. J Biomol NMR 13 289–302, 1999). Extension of the original 20-protein database to 200 proteins increased the fraction of residues for which backbone angles could be predicted from 65 to 74%, while reducing the error rate from 3 to 2.5%. Addition of a two-layer neural network filter to the database fragment selection process forms the basis for a new program, TALOS+, which further enhances the prediction rate to 88.5%, without increasing the error rate. Excluding the 2.5% of residues for which TALOS+ makes predictions that strongly differ from those observed in the crystalline state, the accuracy of predicted ϕ and ψ angles, equals ±13°. Large discrepancies between predictions and crystal structures are primarily limited to loop regions, and for the few cases where multiple X-ray structures are available such residues are often found in different states in the different structures. The TALOS+ output includes predictions for individual residues with missing chemical shifts, and the neural network component of the program also predicts secondary structure with good accuracy.

A structural model for Alzheimer's β-amyloid fibrils based on experimental constraints from solid state NMR

We present a structural model for amyloid fibrils formed by the 40-residue β-amyloid peptide associated with Alzheimers disease (Aβ1–40), based on a set of experimental constraints from solid state NMR spectroscopy. The model additionally incorporates the cross-β structural motif established by x-ray fiber diffraction and satisfies constraints on Aβ1–40 fibril dimensions and mass-per-length determined from electron microscopy. Approximately the first 10 residues of Aβ1–40 are structurally disordered in the fibrils. Residues 12–24 and 30–40 adopt β-strand conformations and form parallel β-sheets through intermolecular hydrogen bonding. Residues 25–29 contain a bend of the peptide backbone that brings the two β-sheets in contact through sidechain-sidechain interactions. A single cross-β unit is then a double-layered β-sheet structure with a hydrophobic core and one hydrophobic face. The only charged sidechains in the core are those of D23 and K28, which form salt bridges. Fibrils with minimum mass-per-length and diameter consist of two cross-β units with their hydrophobic faces juxtaposed.

Consistent blind protein structure generation from NMR chemical shift data

Protein NMR chemical shifts are highly sensitive to local structure. A robust protocol is described that exploits this relation for de novo protein structure generation, using as input experimental parameters the 13Cα, 13Cβ, 13C′, 15N, 1Hα and 1HN NMR chemical shifts. These shifts are generally available at the early stage of the traditional NMR structure determination process, before the collection and analysis of structural restraints. The chemical shift based structure determination protocol uses an empirically optimized procedure to select protein fragments from the Protein Data Bank, in conjunction with the standard ROSETTA Monte Carlo assembly and relaxation methods. Evaluation of 16 proteins, varying in size from 56 to 129 residues, yielded full-atom models that have 0.7–1.8 Å root mean square deviations for the backbone atoms relative to the experimentally determined x-ray or NMR structures. The strategy also has been successfully applied in a blind manner to nine protein targets with molecular masses up to 15.4 kDa, whose conventional NMR structure determination was conducted in parallel by the Northeast Structural Genomics Consortium. This protocol potentially provides a new direction for high-throughput NMR structure determination.

Pulse sequences for removal of the effects of cross correlation between dipolar and chemical-shift anisotropy relaxation mechanisms on the measurement of heteronuclear T1 and T2 values in proteins

The effects of cross correlation between dipolar and chemical-shift anisotropy relaxation interactions on the measurement of heteroatom T1 and T2 relaxation times in proteins is considered. It is shown that such effects can produce errors of approximately 25% in the measurement of 15N transverse relaxation times at a field strength of 11.8 T. Cross correlation has a less significant effect on the measurement of 15N spin-lattice relaxation rates and for proteins the errors in T1 decrease as a function of increasing molecular weight. Nevertheless, for T1 measurements at 11.8 T errors of approximately 15 and 5% are calculated for proteins with correlation times, τc, of 5 and 9 ns, respectively. Pulse sequences which eliminate dipolar and chemical-shift anisotropy cross-correlation effects are described. These sequences are used to make more accurate measurements of 15N T1 and T2 values of staphylococcal nuclease and to determine errors in these parameters that result when cross correlations are present.

Measurement of homo- and heteronuclear J couplings from quantitative J correlation

Publisher Summary This chapter discusses a third method, referred to as quantitative “ J correlation.” In this approach, the J coupling is obtained from the fraction of magnetization that is transferred from a nucleus to its J -coupled partner. Quantitative J correlation is a useful and general approach for measuring a large variety of two- and three-bond homo- and heteronuclear J couplings, providing access to the study of a large number of dihedral angles in proteins. The approach is not limited to isotopically enriched proteins and has also been used to measure a large number of heteronuclear 1 H- 13 C J couplings in the cyclic decapeptide gramicidin S. The quantitative J correlation experiments are particularly useful for defining the X∼ angle, as a substantial number of J couplings defined by this angle can now be measured. The large number of J couplings overdetermines χ l if there exist only a single rotametric state. In cases of rotamer averaging, however, this large number of independent J measurements provides a unique opportunity to characterize the motional averaging process.

Measurement of HN-H? J couplings in calcium-free calmodulin using new 2D and 3D water-flip-back methods

SummaryTwo new methods are described for the measurement of three-bond JHNHαcouplings in proteins isotopically enriched with 15N. Both methods leave the water magnetization in an unsaturated state, parallel to the z-axis, and therefore offer significant enhancements in sensitivity for rapidly exchanging backbone amide protons. The J couplings can be measured either from a set of constant-time 2D 1H-15N HMQC spectra, which are modulated in intensity by JHNHα, or from a water-flip-back version of the 3D HNHA experiment. The method is demonstrated for a sample of calcium-free calmodulin. Residues Lys75-Asp80 have JHNHαvalues in the 6–7 Hz range, suggesting that a break in the ‘central helix’ occurs at the same position as previously observed in solution NMR studies of Ca2+-ligated calmodulin.

Overall structure and sugar dynamics of a DNA dodecamer from homo- and heteronuclear dipolar couplings and 31P chemical shift anisotropy.

The solution structure of d(CGCGAATTCGCG)2 has been determined on the basis of an exceptionally large set of residual dipolar couplings. In addition to the heteronuclear 13C-1H and 15N-1H and qualitative homonuclear 1H-1H dipolar couplings, previously measured in bicelle medium, more than 300 quantitative 1H-1H and 22 31P-1H dipolar restraints were obtained in liquid crystalline Pf1 medium, and 22 31P chemical shift anisotropy restraints. High quality DNA structures can be obtained solely on the basis of these new restraints, and these structures are in close agreement with those calculated previously on the basis of 13C-1H and 15N-1H dipolar couplings. In the newly calculated structures, 31P-1H dipolar and 3JsubH3′Psub couplings and 31P CSA data restrain the phosphodiester backbone torsion angles. The final structure represents a quite regular B-form helix with a modest bending of ∼10°, which is essentially independent of whether or not electrostatic terms are used in the calculation. Combined, the number of homo- and heteronuclear dipolar couplings significantly exceeds the number of degrees of freedom in the system. Results indicate that the dipolar coupling data cannot be fit by a single structure, but are compatible with the presence of rapid equilibria between C2′-endo and C3′-endo deoxyribose puckers (sugar switching). The C2′-H2′/H2′′ dipolar couplings in B-form DNA are particularly sensitive to sugar pucker and yield the largest discrepancies when fit to a single structure. To resolve these discrepancies, we suggest a simplified dipolar coupling analysis that yields N/S equilibria for the ribose sugar puckers, which are in good agreement with previous analyses of NMR JHH couplings, with a population of the minor C3′-endo form higher for pyrimidines than for purines.

Molecular Fragment Replacement Approach to Protein Structure Determination by Chemical Shift and Dipolar Homology Database Mining

A novel approach is described for determining backbone structures of proteins that is based on finding fragments in the protein data bank (PDB). For each fragment in the target protein, usually chosen to be 7-10 residues in length, PDB fragments are selected that best fit to experimentally determined one-bond heteronuclear dipolar couplings and that show agreement between chemical shifts predicted for the PDB fragment and experimental values for the target fragment. These fragments are subsequently refined by simulated annealing to improve agreement with the experimental data. If the lowest-energy refined fragments form a unique structural cluster, this structure is accepted and side chains are added on the basis of a conformational database potential. The sequential backbone assembly process extends the chain by translating an accepted fragment onto it. For several small proteins, with extensive sets of dipolar couplings measured in two alignment media, a unique final structure is obtained that agrees well with structures previously solved by conventional methods. With less dipolar input data, large, oriented fragments of each protein are obtained, but their relative positioning requires either a small set of translationally restraining nuclear Overhauser enhancements (NOEs) or a protocol that optimizes burial of hydrophobic groups and pairing of beta-strands.