Can molecular dynamics simulations provide high‐resolution refinement of protein structure?

Recent advances in efficient and accurate treatment of solvent with the generalized Born approximation (GB) have made it possible to substantially refine the protein structures generated by various prediction tools through detailed molecular dynamics simulations. As demonstrated in a recent CASPR experiment, improvement can be quite reliably achieved when the initial models are sufficiently close to the native basin (e.g., 3-4 A C(alpha) RMSD). A key element to effective refinement is to incorporate reliable structural information into the simulation protocol. Without intimate knowledge of the target and prediction protocol used to generate the initial structural models, it can be assumed that the regular secondary structure elements (helices and strands) and overall fold topology are largely correct to start with, such that the protocol limits itself to the scope of refinement and focuses the sampling in vicinity of the initial structure. The secondary structures can be enforced by dihedral restraints and the topology through structural contacts, implemented as either multiple pair-wise C(alpha) distance restraints or a single sidechain distance matrix restraint. The restraints are weakly imposed with flat-bottom potentials to allow sufficient flexibility for structural rearrangement. Refinement is further facilitated by enhanced sampling of advanced techniques such as the replica exchange method (REX). In general, for single domain proteins of small to medium sizes, 3-5 nanoseconds of REX/GB refinement simulations appear to be sufficient for reasonable convergence. Clustering of the resulting structural ensembles can yield refined models over 1.0 A closer to the native structure in C(alpha) RMSD. Substantial improvement of sidechain contacts and rotamer states can also be achieved in most cases. Additional improvement is possible with longer sampling and knowledge of the robust structural features in the initial models for a given prediction protocol. Nevertheless, limitations still exist in sampling as well as force field accuracy, manifested as difficulty in refinement of long and flexible loops.     

Structure and dynamics of reduced Bacillus pasteurii cytochrome c: oxidation state dependent properties and implications for electron transfer processes

The solution structure of reduced Bacillus pasteurii cytochrome c, which has only 71 amino acids, has been determined by NMR to an RMSD of 0.46 +/- 0.08 A for all backbone atoms and 0.79 +/- 0.08 A for all heavy atoms and refined through restrained energy minimization. The target function out of 1645 constraints is 0.52 +/- 0.11 A(2), and the penalty function is 66 +/- 12 kJ mol(-)(1). The structure appears very similar to that in the oxidized state, only Trp87 and the propionates showing significant differences. The mobility was investigated through (15)N R(1) and R(2) relaxation rates, (15)N-(1)H NOE, and (1)H/(2)H exchange. It is found that the oxidized form is generally more mobile than the reduced one. By comparing the redox-state dependence of the structural/dynamic properties of Fe-S proteins, cytochrome c, and blue copper proteins, hints are provided for a better comprehension of the electron transfer processes.     

TASSER-based refinement of NMR structures

The TASSER structure prediction algorithm is employed to investigate whether NMR structures can be moved closer to their corresponding X-ray counterparts by automatic refinement procedures. The benchmark protein dataset includes 61 nonhomologous proteins whose structures have been determined by both NMR and X-ray experiments. Interestingly, by starting from NMR structures, the majority (79%) of TASSER refined models show a structural shift toward their X-ray structures. On average, the TASSER refined models have a root-mean-square-deviation (RMSD) from the X-ray structure of 1.785 A (1.556 A) over the entire chain (aligned region), while the average RMSD between NMR and X-ray structures (RMSD(NMR_X-ray)) is 2.080 A (1.731 A). For all proteins having a RMSD(NMR_X-ray) >2 A, the TASSER refined structures show consistent improvement. However, for the 34 proteins with a RMSD(NMR_X-ray) <2 A, there are only 21 cases (60%) where the TASSER model is closer to the X-ray structure than NMR, which may be due to the inherent resolution of TASSER. We also compare the TASSER models with 12 NMR models in the RECOORD database that have been recalculated recently by Nederveen et al. from original NMR restraints using the newest molecular dynamics tools. In 8 of 12 cases, TASSER models show a smaller RMSD to X-ray structures; in 3 of 12 cases, where RMSD(NMR_X-ray) <1 A, RECOORD does better than TASSER. These results suggest that TASSER can be a useful tool to improve the quality of NMR structures.     

On the molecular basis of the recognition of angiotensin II (AII). NMR structure of AII in solution compared with the X-ray structure of AII bound to the mAb Fab131.

The high-resolution 3D structure of the octapeptide hormone angiotensin II (AII) in aqueous solution has been obtained by simulated annealing calculations, using high-resolution NMR-derived restraints. After final refinement in explicit water, a family of 13 structures was obtained with a backbone RMSD of 0.73 +/- 0.23 A. AII adopts a fairly compact folded structure, with its C-terminus and N-terminus approaching to within approximately 7.2 A of each other. The side chains of Arg2, Tyr4, Ile5 and His6 are oriented on one side of a plane defined by the peptide backbone, and the Val3 and Pro7 are pointing in opposite directions. The stabilization of the folded conformation can be explained by the stacking of the Val3 side chain with the Pro7 ring and by a hydrophobic cluster formed by the Tyr4, Ile5 and His6 side chains. Comparison between the NMR-derived structure of AII in aqueous solution and the refined crystal structure of the complex of AII with a high-affinity mAb (Fab131) [Garcia, K.C., Ronco, P.M., Verroust, P.J., Brunger, A.T., Amzel, L.M. (1992) Science257, 502-507] provides important quantitative information on two common structural features: (a) a U-shaped structure of the Tyr4-Ile5-His6-Pro7 sequence, which is the most immunogenic epitope of the peptide, with the Asp1 side chain oriented towards the interior of the turn approaching the C-terminus; (b) an Asx-turn-like motif with the side chain aspartate carboxyl group hydrogen-bonded to the main chain NH group of Arg2. It can be concluded that small rearrangements of the epitope 4-7 in the solution structure of AII are required by a mean value of 0.76 +/- 0.03 A for structure alignment and approximately 1.27 +/- 0.02 A for sequence alignment with the X-ray structure of AII bound to the mAb Fab131. These data are interpreted in terms of a biological "nucleus" conformation of the hormone in solution, which requires a limited number of structural rearrangements for receptor-antigen recognition and binding.     

Improving NMR protein structure quality by Rosetta refinement: a molecular replacement study

The structure of human protein HSPC034 has been determined by both solution nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. Refinement of the NMR structure ensemble, using a Rosetta protocol in the absence of NMR restraints, resulted in significant improvements not only in structure quality, but also in molecular replacement (MR) performance with the raw X-ray diffraction data using MOLREP and Phaser. This method has recently been shown to be generally applicable with improved MR performance demonstrated for eight NMR structures refined using Rosetta (Qian et al., Nature 2007;450:259-264). Additionally, NMR structures of HSPC034 calculated by standard methods that include NMR restraints have improvements in the RMSD to the crystal structure and MR performance in the order DYANA, CYANA, XPLOR-NIH, and CNS with explicit water refinement (CNSw). Further Rosetta refinement of the CNSw structures, perhaps due to more thorough conformational sampling and/or a superior force field, was capable of finding alternative low energy protein conformations that were equally consistent with the NMR data according to the Recall, Precision, and F-measure (RPF) scores. On further examination, the additional MR-performance shortfall for NMR refined structures as compared with the X-ray structure were attributed, in part, to crystal-packing effects, real structural differences, and inferior hydrogen bonding in the NMR structures. A good correlation between a decrease in the number of buried unsatisfied hydrogen-bond donors and improved MR performance demonstrates the importance of hydrogen-bond terms in the force field for improving NMR structures. The superior hydrogen-bond network in Rosetta-refined structures demonstrates that correct identification of hydrogen bonds should be a critical goal of NMR structure refinement. Inclusion of nonbivalent hydrogen bonds identified from Rosetta structures as additional restraints in the structure calculation results in NMR structures with improved MR performance.     

Monitoring the early steps of unfolding of dicalcium and mono-Ce3+-substituted forms of P43M calbindin D9k

Early steps of unfolding of P43M Calbindin D(9k) have been evaluated by NMR spectroscopy on the native dicalcium and on the paramagnetic monocerium-substituted derivative. Although at 2 M GdmHCl the protein core maintains its overall folding and structure, amide (15)N R(2) measurements and cross correlation rates between N-H dipole-dipole relaxation and (15)N CSA relaxation reveal a closer and stronger packing of the hydrophobic interactions in the protein as a response to the presence of denaturing agents in solution. A complete reorientation of the Met43 side chain toward the hydrophobic core is accomplished by the disappearance of the millisecond dynamics observed on the native form of Calbindin D(9k), while cross correlation rates provide evidence that the two-way hydrogen bond between Leu23 and Val61 is broken or substantially weakened. The substitution of the calcium ion in site II with the paramagnetic Ce(3+) ion allowed us to obtain a number of long-range nonconventional constraints, namely, pseudocontact shifts, which were used, together with the NOEs collected on the native state, to monitor subtle structural variations occurring in the non-native state of the protein. Although the average rmsd between the structures of native and non-native states is small (0.48 A), structural rearrangements could be reliably identified. Our results provide unprecedented information about the behavior of Calbindin D(9k) during the early steps of unfolding. Furthermore, they constitute strong evidence of the efficiency of paramagnetism-based constraints in monitoring subtle structural changes that are beyond the sensitivity of an approach based only on NOE.     

Cobalt hexammine inhibition of the hammerhead ribozyme

The effects of Co(NH(3))(6)(3+) on the hammerhead ribozyme are analyzed using several techniques, including activity measurements, electron paramagnetic resonance (EPR), and circular dichroism (CD) spectroscopies and thermal denaturation studies. Co(NH(3))(6)(3+) efficiently displaces Mn(2+) bound to the ribozyme with an apparent dissociation constant of K(d app) = 22 +/- 4.2 microM in 500 microM Mn(2+) (0.1 M NaCl). Displacement of Mn(2+) coincides with Co(NH(3))(6)(3+) inhibition of hammerhead activity in 500 microM Mn(2+), reducing the activity of the WT hammerhead by approximately 15-fold with an inhibition constant of K(i) = 30.9 +/- 2.3 microM. A residual 'slow' activity is observed in the presence of Co(NH(3))(6)(3+) and low concentrations of Mn(2+). Under these conditions, a single Mn(2+) ion remains bound and has a low-temperature EPR spectrum identical to that observed previously for the highest affinity Mn(2+) site in the hammerhead ribozyme in 1 M NaCl, tentatively attributed to the A9/G10.1 site [Morrissey, S. R. , Horton, T. E., and DeRose, V. J. (2000) J. Am. Chem. Soc. 122, 3473-3481]. Circular dichroism and thermal denaturation experiments also reveal structural effects that accompany the observed inhibition of cleavage and Mn(2+) displacement induced by addition of Co(NH(3))(6)(3+). Taken together, the data indicate that a high-affinity Co(NH(3))(6)(3+) site is responsible for significant inhibition accompanied by structural changes in the hammerhead ribozyme. In addition, the results support a model in which at least two types of metal sites, one of which requires inner-sphere coordination, support hammerhead activity.     

Crystal structure of guanosine-free ribonuclease T1, complexed with vanadate (V), suggests conformational change upon substrate binding

Ribonuclease T1 was crystallized in the presence of vanadate(V). The crystal structure was solved by molecular replacement and refined by least-squares methods using stereochemical restraints. The refinement was based on data between 10 and 1.8 A and converged at a crystallographic R factor of 0.137. Except for the substrate-recognition site the three-dimensional structure of ribonuclease T1 closely resembles the structure of the enzyme complexed with guanosine 2'-phosphate and its derivatives. A tetrahedral anion was found at the catalytic site and identified as H2VO4-. This is the first crystal structure of ribonuclease T1 determined in the absence of bound substrate analogue. Distinct structural differences between guanosine-free and complexed ribonuclease T1 are observed at the base-recognition site: The side chains of Tyr45 and Glu46 and the region around Asn98 changed their conformations, and the peptide bond between Asn43 and Asn44 has turned around by 140 degrees. We suggest that the structural differences seen in the crystal structures of free and complexed ribonuclease T1 are related to conformational adjustments associated with the substrate binding process.     

A refined model for the solution structure of oxidized putidaredoxin

A refined model for the solution structure of oxidized putidaredoxin (Pdxo), a Cys4Fe2S2 ferredoxin, has been determined. A previous structure (Pochapsky et al. (1994) Biochemistry 33, 6424-6432; PDB entry ) was calculated using the results of homonuclear two-dimensional NMR experiments. New data has made it possible to calculate a refinement of the original Pdxo solution structure. First, essentially complete assignments for diamagnetic 15N and 13C resonances of Pdxo have been made using multidimensional NMR methods, and 15N- and 13C-resolved NOESY experiments have permitted the identification of many new NOE restraints for structural calculations. Stereospecific assignments for leucine and valine CH3 resonances were made using biosynthetically directed fractional 13C labeling, improving the precision of NOE restraints involving these residues. Backbone dihedral angle restraints have been obtained using a combination of two-dimensional J-modulated 15N,1H HSQC and 3D (HN)CO(CO)NH experiments. Second, the solution structure of a diamagnetic form of Pdx, that of the C85S variant of gallium putidaredoxin, in which a nonligand Cys is replaced by Ser, has been determined (Pochapsky et al. (1998) J. Biomol. NMR 12, 407-415), providing information concerning structural features not observable in the native ferredoxin due to paramagnetism. Third, a crystal structure of a closely related ferredoxin, bovine adrenodoxin, has been published (Müller et al. (1998) Structure 6, 269-280). This structure has been used to model the metal binding site structure in Pdx. A family of fourteen structures is presented that exhibits an rmsd of 0.51 A for backbone heavy atoms and 0.83 A for all heavy atoms. Exclusion of the modeled metal binding loop region reduces overall the rmsd to 0.30 A for backbone atoms and 0.71 A for all heavy atoms.     

Metropolis Monte Carlo calculations of DNA structure using internal coordinates and NMR distance restraints: An alternative method for generating a high-resolution solution structure

Summary A new method, a restrained Monte Carlo (rMC) calculation, is demonstrated for generating high-resolution structures of DNA oligonucleotides in solution from interproton distance restraints and bounds derived from complete relaxation matrix analysis of two-dimensional nuclear Overhauser effect (NOE) spectral peak intensities. As in the case of restrained molecular dynamics (rMD) refinement of structures, the experimental distance restraints and bounds are incorporated as a pseudo-energy term (or penalty function) into the mathematical expression for the molecular energy. However, the use of generalized helical parameters, rather than Cartesian coordinates, to define DNA conformation increases efficiency by decreasing by an order of magnitude the number of parameters needed to describe a conformation and by simplifying the potential energy profile. The Metropolis Monte Carlo method is employed to simulate an annealing process. The rMC method was applied to experimental 2D NOE data from the octamer duplex d(GTA-TAATG)·d(CATTATAC). Using starting structures from different locations in conformational space (e.g. A-DNA and B-DNA), the rMC calculations readily converged, with a root-mean-square deviation (RMSD) of <0.3 Å between structures generated using different protocols and starting structures. Theoretical 2D NOE peak intensities were calculated for the rMC-generated structures using the complete relaxation matrix program CORMA, enabling a comparison with experimental intensities via residual indices. Simulation of the vicinal proton coupling constants was carried out for the structures generated, enabling a comparison with the experimental deoxyribose ring coupling constants, which were not utilized in the structure determination in the case of the rMC simulations. Agreement with experimental 2D NOE and scalar coupling data was good in all cases. The rMC structures are quite similar to that refined by a traditional restrained MD approach (RMSD<0.5 Å) despite the different force fields used and despite the fact that MD refinement was conducted with additional restraints imposed on the endocyclic torsion angles of deoxyriboses. The computational time required for the rMC and rMD calculations is about the same. A comparison of structural parameters is made and some limitations of both methods are discussed with regard to the average nature of the experimental restraints used in the refinement.Abbreviations MC Monte Carlo - rMC restrained Monte Carlo - MD molecular dynamics - rMD restrained molecular dynamics - DG distance geometry - EM energy minimization - 2D NOE two-dimensional nuclear Overhauser effect - DQF-COSY double-quantum-filtered correlation spectroscopy - RMSD root-mean-square deviation To whom correspondence should be addressed.     

Solution structure and NH exchange studies of the MutT pyrophosphohydrolase complexed with Mg(2+) and 8-oxo-dGMP,a tightly bound product

To learn the structural basis for the unusually tight binding of 8-oxo-nucleotides to the MutT pyrophosphohydrolase of Escherichia coli (129 residues), the solution structure of the MutT-Mg(2+)-8-oxo-dGMP product complex (K(D) = 52 nM) was determined by standard 3-D heteronuclear NMR methods. Using 1746 NOEs (13.5 NOEs/residue) and 186 phi and psi values derived from backbone (15)N, Calpha, Halpha, and Cbeta chemical shifts, 20 converged structures were computed with NOE violations 相似文献   

A Refined Model for the Structure of Acireductone Dioxygenase from Klebsiella ATCC 8724 Incorporating Residual Dipolar Couplings

Acireductone dioxygenase (ARD) from Klebsiella ATCC 8724 is a metalloenzyme that is capable of catalyzing different reactions with the same substrates (acireductone and O₂) depending upon the metal bound in the active site. A model for the solution structure of the paramagnetic Ni²⁺-containing ARD has been refined using residual dipolar couplings (RDCs) measured in two media. Additional dihedral restraints based on chemical shift (TALOS) were included in the refinement, and backbone structure in the vicinity of the active site was modeled from a crystallographic structure of the mouse homolog of ARD. The incorporation of residual dipolar couplings into the structural refinement alters the relative orientations of several structural features significantly, and improves local secondary structure determination. Comparisons between the solution structures obtained with and without RDCs are made, and structural similarities and differences between mouse and bacterial enzymes are described. Finally, the biological significance of these differences is considered.     

Strategies for the uses of lanthanide NMR shift probes in the determination of protein structure in solutio. Application to the EF calcium binding site of carp parvalbumin.

The homologous sequences observed for many calcium binding proteins such as parvalbumin, troponin C, the myosin light chains, and calmodulin has lead to the hypothesis that these proteins have homologous structures at the level of their calcium binding sites. This paper discusses the development of a nuclear magnetic resonance (NMR) technique which will enable us to test this structural hypothesis in solution. The technique involves the substitution of a paramagnetic lanthanide ion for the calcium ion which results in lanthanide induced shifts and broadening in the 1H NMR spectrum of the protein. These shifts are sensitive monitors of the precise geometrical orientation of each proton nucleus relative to the metal. The values of several parameters in the equation relating the NMR shifts to the structure are however known as priori. We have attempted to determine these parameters, the orientation and principal elements of the magnetic susceptibility tensor of the protein bound metal, by studying the lanthanide induced shifts for the protein parvalbumin whose structure has been determined by x-ray crystallographic techniques. The interaction of the lanthanide ytterbium with parvalbumin results in high resolution NMR spectra exhibiting a series of resonances with shifts spread over the range 32 to -19 ppm. The orientation and principal elements of the ytterbium magnetic susceptibility tensor have been determined using three assigned NMR resonances, the His-26 C2 and C4 protons and the amino terminal acetyl protons, and seven methyl groups; all with known geometry relative to the EF calcium binding site. The elucidation of these parameters has allowed us to compare the observed spectrum of the nuclei surrounding the EF calcium binding site of parvalbumin with that calculated from the x-ray structure. A significant number of the calculated shifts are larger than any of the observed shifts. We feel that a refinement of the x-ray based proton coordinates will be possible utilizing the geometric information contained in the lanthanide shifted NMR spectrum.     

Structural analysis of Mg2+ and Ca2+ binding, myristoylation, and dimerization of the neuronal calcium sensor and visinin-like protein 1 (VILIP-1)

Visinin-like protein 1 (VILIP-1) belongs to the neuronal calcium sensor family of Ca(2+)-myristoyl switch proteins that regulate signal transduction in the brain and retina. Here we analyze Ca(2+) and Mg(2+) binding, characterize metal-induced conformational changes, and determine structural effects of myristoylation and dimerization. Mg(2+) binds functionally to VILIP-1 at EF3 (ΔH = +1.8 kcal/mol and K(D) = 20 μM). Unmyristoylated VILIP-1 binds two Ca(2+) sequentially at EF2 and EF3 (K(EF3) = 0.1 μM and K(EF2) = 1-4 μM), whereas myristoylated VILIP-1 binds two Ca(2+) with lower affinity (K(D) = 1.2 μM) and positive cooperativity (Hill slope = 1.5). NMR assignments and structural analysis indicate that Ca(2+)-free VILIP-1 contains a sequestered myristoyl group like that of recoverin. NMR resonances of the attached myristate exhibit Ca(2+)-dependent chemical shifts and NOE patterns consistent with Ca(2+)-induced extrusion of the myristate. VILIP-1 forms a dimer in solution independent of Ca(2+) and myristoylation. The dimerization site is composed of residues in EF4 and the loop region between EF3 and EF4, confirmed by mutagenesis. We present the structure of the VILIP-1 dimer and a Ca(2+)-myristoyl switch to provide structural insights into Ca(2+)-induced trafficking of nicotinic acetylcholine receptors.     

Are time-averaged restraints necessary for nuclear magnetic resonance refinement? A model study for DNA.

A recently suggested method for refinement of structural data obtained from two-dimensional nuclear magnetic resonance experiments using molecular dynamics (MD) is explored. In this method, the time-averaged values of the appropriate internal co-ordinates of the molecule, calculated from the MD trajectory, are driven by restraints towards the experimental target values. This contrasts with most refinement procedures currently in use, where restraints are applied based on the instantaneous values of the appropriate co-ordinates. Both refinement methods are applied to the EcoRI restriction site DNA hexamer d(GAATTC)2, using target nuclear Overhauser enhancement distances derived from a one nanosecond unrestrained MD simulation of this structure. The resulting refined structures are compared to the results of the unrestrained MD trajectory, which serves as our "experimental" data. We show that although both methods can yield an average structure with the correct gross morphology, the new method allows both a much more realistic picture of inherent flexibility, and reproduces fine conformational detail better, such as sequence dependency. We also analyze the very long MD trajectory generated here (longer than any previously reported for a DNA oligomer), and find that significantly shorter simulations, typical of those frequently performed, may not yield acceptably reliable values for certain structural parameters.     

NMR-validated structural model for oxidized<Emphasis Type="Italic"> Rhodopseudomonas palustris</Emphasis> cytochrome<Emphasis Type="Italic"> c</Emphasis><Subscript>556</Subscript>

The structure of oxidized Rhodopseudomonas palustris cytochrome c ₅₅₆ has been modeled after that of high-spin cytochrome c from the same bacterium, the latter being the protein with the greatest sequence identity (35%) among all sequenced proteins in the genomes. The two proteins differ in the number of ligands to iron and in spin state, the former being six-coordinate low-spin and the latter five-coordinate high-spin. In order to validate this modeled structure, several structural restraints were obtained by performing a restricted set of NMR experiments, without performing a complete assignment of the protein signals. The aim was to exploit the special restraints arising from the paramagnetism of the metal ion. A total of 43 residual-dipolar-coupling and 74 pseudocontact-shift restraints, which together sampled all regions of the protein, were used in conjunction with over 40 routinely obtained NOE distance restraints. A calculation procedure was undertaken combining the program MODELLER and the solution structure determination program PARAMAGNETIC DYANA, which includes paramagnetism-based restraints. The directions and magnitude of the magnetic susceptibility anisotropy tensor were also calculated. The approach readily provides useful results, especially for paramagnetic metalloproteins of moderate to large dimensions.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-003-0511-2     

Localized solution structure refinement of an F45W variant of ubiquitin using stochastic boundary molecular dynamics and NMR distance restraints.

The local structure within an 8-A radius around residue 45 of a recombinant F45W variant of human ubiquitin has been determined using 67 interproton distance restraints measured by two-dimensional proton NMR. Proton chemical shift evidence indicates that structural perturbations due to the F45W mutation are minimal and limited to the immediate vicinity of the site of mutation. Simulated annealing implemented with stochastic boundary molecular dynamics was applied to refine the structure of Trp 45 and 10 neighboring residues. The stochastic boundary method allowed the entire protein to be reassembled from the refined coordinates and the outlying unrefined coordinates with little distortion at the boundary. Refinement began with four low-energy indole ring orientations of F45W-substituted wild-type (WT) ubiquitin crystal coordinates. Distance restraints were derived from mostly long-range NOE cross peaks with 51 restraints involving the Trp 45 indole ring. Tandem refinements of 64 structures were done using either (1) upper and lower bounds derived from qualitative inspection of NOE crosspeak intensities or (2) quantitative analysis of cross-peak heights using the program MARDIGRAS. Though similar to those based on qualitative restraint, structures obtained using quantitative NOE analysis were superior in terms of precision and accuracy as measured by back-calculated sixth-root R factors. The six-membered portion of the indole ring is nearly coincident with the phenyl ring of the WT and the indole NH is exposed to solvent. Accommodation of the larger ring is accompanied by small perturbations in the backbone and a 120 degrees rotation of the chi 2 dihedral angle of Leu 50.     

Paramagnetism-Based Restraints for Xplor-NIH

Modules that use paramagnetism-based NMR restraints have been developed and integrated in the well known program for solution structure determination Xplor-NIH; the complete set of such modules is called PARArestraints for Xplor-NIH. Paramagnetism-based restraints are paramagnetic relaxation enhancements, pseudocontact shifts, residual dipolar couplings due to metal and overall magnetic anisotropy, and cross correlation between Curie relaxation and nuclear-nuclear dipolar relaxation. The complete program has been tested by back-calculating NOEs and paramagnetism-based restraints from the X-ray structure of cytochrome c (553) from B. pasteurii. Furthermore, the same experimental restraints previously used to determine the solution structure of cytochrome c (553) itself, of cytochrome b (5), and of calbindin D(9k) with the program PARAMAGNETIC DYANA, have been used for structure calculations by using PARArestraints for Xplor-NIH. The agreement between the two programs is quite satisfactory and validates both protocols.     

Molecular dynamics re-refinement of two different small RNA loop structures using the original NMR data suggest a common structure

Restrained molecular dynamics simulations are a robust, though perhaps underused, tool for the end-stage refinement of biomolecular structures. We demonstrate their utility-using modern simulation protocols, optimized force fields, and inclusion of explicit solvent and mobile counterions-by re-investigating the solution structures of two RNA hairpins that had previously been refined using conventional techniques. The structures, both domain 5 group II intron ribozymes from yeast ai5γ and Pylaiella littoralis, share a nearly identical primary sequence yet the published 3D structures appear quite different. Relatively long restrained MD simulations using the original NMR restraint data identified the presence of a small set of violated distance restraints in one structure and a possibly incorrect trapped bulge nucleotide conformation in the other structure. The removal of problematic distance restraints and the addition of a heating step yielded representative ensembles with very similar 3D structures and much lower pairwise RMSD values. Analysis of ion density during the restrained simulations helped to explain chemical shift perturbation data published previously. These results suggest that restrained MD simulations, with proper caution, can be used to "update" older structures or aid in the refinement of new structures that lack sufficient experimental data to produce a high quality result. Notable cautions include the need for sufficient sampling, awareness of potential force field bias (such as small angle deviations with the current AMBER force fields), and a proper balance between the various restraint weights.