The influence of helix morphology on co-operative polyamide backbone conformational flexibility in peptide nucleic acid complexes.

A systematic analysis of peptide nucleic acid (PNA) complexes deposited in the Protein Data Bank has been carried out using a set of contiguous atom torsion angle definitions. The analysis is complemented by molecular mechanics adiabatic potential energy calculations on hybrid PNA-nucleic acid model systems. Hitherto unobserved correlations in the values of the (alpha and epsilon) dihedral angles flanking the backbone secondary amide bond are found. This dihedral coupling forms the basis of a PNA backbone conformation classification scheme. Six conformations are thus characterised in experimental structures. Helix morphology is found to exert a significant influence on backbone conformation and flexibility: Watson-Crick PNA strands in complexes with DNA and RNA, that possess A-like base-pair stacking, adopt backbone conformations distinct from those in PNA.DNA-PNA triplex and PNA-PNA duplex P-helix forms. Solvation effects on Watson-Crick PNA backbone conformation in heterotriplexes are discussed and the possible involvement of inter-conformational transitions and dihedral angle uncoupling in asymmetric heteroduplex base-pair breathing is suggested.     

Local deformation studies of chain molecules: differential conditions for changes of dihedral angles

New first and second-order differential equations for changes of dihedral angles characterizing local deformations of chain molecules with fixed bond lengths and bond angles are derived. Two methods for integrating the differential relations are given. The proposed method is used to generate a path of locally deformed conformations around a β-turn region of a small protein, bovine pancreatic trypsin inhibitor. The variable regions change their conformations by more than 3 Å root-mean-square distance value whereas the fixed regions stay within 0.02 Å. Possible applications of this method are in the field of computer graphics, Monte Carlo simulations, and energy minimization calculations of chain molecules.     

Preferred conformation of the benzyloxycarbonyl-amino group in peptides

Structural parameters, derived from X-ray crystallographic data, have been compiled for 35 derivatives of amino acids, peptides, and related compounds, which contain the N-terminal benzyloxycarbonyl (Z) group. The geometry of the urethane moiety of this end group is closely similar to that of the tert-butoxycarbonyl (Boc) group, except for a relaxation of some bond angles because the Z group is sterically less crowded than the Boc group. For the same reason, the Z group has greater conformational flexibility. As a result, packing forces in the crystal may cause greater deformations of bond angles, resulting in larger variations of observed bond lengths and bond angles than in Boc-peptide crystals. The aromatic rings of the Z end groups tend to stack in crystals. Conformational energy calculations indicate that most conformations of Z-amino acid-N'-methylamides and of corresponding Boc derivatives have similar dihedral angles and relative energies, i.e. the nature of the N-terminal end group has little effect on the conformational preferences of the residue next to it. In particular, the computed fraction of molecules with a cis urethane (C-N) bond is similar for the two derivatives: 0.51 and 0.42 in Boc-Pro-NHCH3 and Z-Pro-NHCH3, respectively, and 0.02 in the two Ala derivatives. There exist several computed conformations of Z-Ala-NHCH3 and Z-Pro-NHCH3 in which the phenyl ring and the C-terminal methylamide group are close to each other. Because of favorable nonbonded interactions, such conformations are of low energy.     

Conformational studies of somatostatin and selected analogues by Raman spectroscopy

Laser Raman spectroscopy has been used to study the conformations of somatostatin and some selected analogues in aqueous solution. The results indicate that the CS-SC dihedral angles of somatostatin and of these analogues (except [Ala3,14]-SS, which has no disulfide bond) are within 20 degrees of +/- 85 degrees, and the SS-CC dihedral angles are predominantly in the range of 50 degrees-180 degrees. Furthermore, from the behavior of the amide I' and amide III bands, it appears that somatostatin adopts a beta-pleated sheet structure, whereas its analogues are less ordered (to varying degrees).     

Flexibility of bovine pancreatic trypsin inhibitor

The native conformation of a protein may be expressed in terms of the dihedral angles, phi's and psi's for the backbone, and kappa's for the side chains, for a given geometry (bond lengths and bond angles). We have developed a method to obtain the dihedral angles for a low-energy structure of a protein, starting with the X-ray structure; it is applied here to examine the degree of flexibility of bovine pancreatic trypsin inhibitor. Minimization of the total energy of the inhibitor (including nonbonded, electrostatic, torsional, hydrogen bonding, and disulfide loop energies) yields a conformation having a total energy of -221 kcal/mol and a root mean square deviation between all atoms of the computed and experimental structures of 0.63 A. The optimal conformation is not unique, however, there being at least two other conformations of low-energy (-222 and -220 kcal/mol), which resemble the experimental one (root mean square deviations of 0.66 and 0.64 A, respectively). These three conformations are located in different positions in phi, psi space, i.e., with a total deviation of 81 degrees, 100 degrees and 55 degrees from each other (with a root mean square deviation of several degrees per dihedral angle from each other). The nonbonded energies of the backbones, calculated along lines in phi, psi space connecting these three conformations, are all negative, without any intervening energy barriers (on an energy contour map in the phi, psi plane). Side chains were attached at several representative positions in this plane, and the total energy was minimized by varying the kappa's. The energies were of approximately the same magnitude as the previous ones, indicating that the conformation of low energy is flexible to some extent in a restricted region of phi, psi space. Interestingly, the difference delta phi i+1 in phi i+1 for the (i + 1)th residue from one conformation to another is approximately the same as -delta psi i for the ith residue; i.e., the plane of the peptide group between the ith and (i + 1)th residues re-orient without significant changes in the positions of the other atoms. The flexibility of the orientations of the planes of the peptide groups is probably coupled in a cooperative manner to the flexibility of the positions of the backbone and side-chain atoms.     

Consistent force field calculations on 2,5-diketopiperazine and its 3.6-dimethyl derivatives

The minimum energy conformations are calculated for 2, 5-diketopiperazine (DKP) and its 3,6-dimethyl derivatives (DL-DMDKP and LL-DMDKP), using a consistent force field approach developed previously. The energy function parameters that were not required in earlier calculations on alkanes, amides, mid lactams are fitted to spectral and conformational data on the diketopiperazines. Vibrational assignments are suggested for DKP. Conformational energies are also determined over a range of selected values for ring dihedral angles, and the shape of the potential energy functions is examined over deviations from planarity. DKP and LL-DMDKP are found to have non-planar minimum energy conformations, separated from planar by less than a kcal/mole. DL-DMKP exhibits a nearly flat trough about the planar conformation. Calculations of minimum energies with one dihedral angle coordinate constrainted show a coupling between bond angles and dihedral angles in agreement with recent suggestions of Benedetti.     

Extending the accuracy limits of prediction for side-chain conformations

Current techniques for the prediction of side-chain conformations on a fixed backbone have an accuracy limit of about 1.0-1.5 A rmsd for core residues. We have carried out a detailed and systematic analysis of the factors that influence the prediction of side-chain conformation and, on this basis, have succeeded in extending the limits of side-chain prediction for core residues to about 0.7 A rmsd from native, and 94 % and 89 % of chi(1) and chi(1+2 ) dihedral angles correctly predicted to within 20 degrees of native, respectively. These results are obtained using a force-field that accounts for only van der Waals interactions and torsional potentials. Prediction accuracy is strongly dependent on the rotamer library used. That is, a complete and detailed rotamer library is essential. The greatest accuracy was obtained with an extensive rotamer library, containing over 7560 members, in which bond lengths and bond angles were taken from the database rather than simply assuming idealized values. Perhaps the most surprising finding is that the combinatorial problem normally associated with the prediction of the side-chain conformation does not appear to be important. This conclusion is based on the fact that the prediction of the conformation of a single side-chain with all others fixed in their native conformations is only slightly more accurate than the simultaneous prediction of all side-chain dihedral angles.     

Interactions study between the copper II ion and constitutive elements of chitosan structure by DFT calculation

Molecular modeling is particularly useful to understand interactions between various kinds of molecules and ions. This study is aimed at studying the interactions between one Cu(2+) ion and one or several glucosamine residues. The geometries and the interaction energies of all of the complexes involving all of the dimers obtained from glucosamine and N-acetylglucosamine were computed by means of density functional theory (DFT) methods. In a first step, for the two dimers A-A and A-B (A for glucosamine and B for N-acetyl glucosamine), a starting geometry was built, and the energies were calculated using a rigid rotation of 30 degrees intervals for each of the dihedral angles (Phi and Psi) of the glycosidic bond, spanning the whole angular range. These calculations allowed us to retrieve the minimal energy conformation and investigate all possible conformations. The results were compared to some experimental data. In a second step, we investigated the interactions of Cu(2+) with the different possible coordination sites of A. For all complexes considered, the Cu(2+) site was completed with H(2)O and/or OH(-) ligands to have a global neutral charge. The calculations confirmed that the most stable interactions involved the free amino site in a "pending complex". Another pending form was possible considering the participation of the heterocyclic O site, but the latter was less favored. On the other hand, we also showed that glucosamine could not act as a bidentate ligand and that N-acetyl glucosamine was not coordinating with Cu(2+). Finally, our results evidenced a cooperative fixation of Cu(2+) ions when considering the complexation of two successive metal ions on the two consecutive glucosamine residues of the dimer A-A.     

Refined structure of glutathione reductase at 1.54 A resolution

The crystal structure of human glutathione reductase has been established at 1.54 A resolution using a restrained least-squares refinement method. Based on 77,690 independent reflections of better than 10 A resolution, a final R-factor of 18.6% was obtained with a model obeying standard geometry within 0.025 A in bond lengths and 2.4 degrees in bond angles. The final 2Fo-Fc electron density map allows for the distinction of carbon, nitrogen and oxygen atoms with temperature factors below about 25 A2. Apart from 461 amino acid residues and the prosthetic group FAD, the model contains 524 solvent molecules, about 118 of which can be considered an integral part of the enzyme. The largest solvent cluster is at the dimer interface and contains 104 interconnected solvent molecules, part of which are organized in a warped sheet-like structure. The main-chain dihedral angles are well-concentrated in the allowed regions of the Ramachandran plot. The spread of dihedral angles in beta-pleated sheets is much larger than in alpha-helices and especially in alpha-helix cores, indicating the higher plasticity of beta-structures. The analysis revealed a large amount of 3(10)-helix. The side-chain conformations cluster at the staggered positions, and show well-defined preferences. Also, a mobility gradient is observed for side-chains. Non-polar and polar side-chains show average temperature factor increases per bond of 10% and 25%, respectively. A number of alternative conformations of internal side-chains, in particular serines and methionines, have been detected. The extended FAD molecule also shows a mobility gradient between the very rigid flavin (mean value of B) = 8.7 A2) and the more mobile adenine (mean value of B = 16.2 A2). The entire active center is particularly well ordered, with temperature factors around 10 A2. The dimer interface consists of a rigid contact area, which is well conserved in the Escherichia coli enzyme, and a flexible area that is not. Altogether, the buried surfaces at the crystal contacts are half as large as at the dimer interface, but less specific. The refined structure shows clearly that there are no buried cations compensating the charge of the pyrophosphate moiety of FAD. The flavin deviates slightly from standard geometry, which is possibly caused by the polypeptide environment. In contrast to an earlier interpretation, atom N5 of the flavin can accommodate a proton, and it is conceivable that this proton proceeds to the redox-active disulfide.(ABSTRACT TRUNCATED AT 400 WORDS)     

Quantum chemical studies on the conformational structure of bacterial peptidoglycan. II. PCILO calculations on the mono-saccharides

Conformational energy calculations were performed on mono-saccharides of the glycan moiety of bacterial peptidoglycan using PCILO semiempirical quantum chemical method to re-examine the preferred conformations of these molecules so far reported in the empirical studies. The PCILO dihedral angles for some side groups are different from those found in MNDO studies. However, in almost every case the PCILO and empirical calculations suggest the same orientations for the different side groups. Moreover, the strengths of the hydrogen bonds between several groups found in the PCILO calculations are more than those of the empirical and MNDO results.     

The crystal structures of the complexes between bovine beta-trypsin and ten P1 variants of BPTI

The high-resolution X-ray structures have been determined for ten complexes formed between bovine beta-trypsin and P1 variants (Gly, Asp, Glu, Gln, Thr, Met, Lys, His, Phe, Trp) of bovine pancreatic trypsin inhibitor (BPTI). All the complexes were crystallised from the same conditions. The structures of the P1 variants Asp, Glu, Gln and Thr, are reported here for the first time in complex with any serine proteinase. The resolution of the structures ranged from 1.75 to 2.05 A and the R-factors were about 19-20 %. The association constants of the mutants ranged from 1.5x10(4) to 1.7x10(13) M-1. All the structures could be fitted into well-defined electron density, and all had very similar global conformations. All the P1 mutant side-chains could be accomodated at the primary binding site, but relative to the P1 Lys, there were small local changes within the P1-S1 interaction site. These comprised: (1) changes in the number and dynamics of water molecules inside the pocket; (2) multiple conformations and non-optimal dihedral angles for some of the P1 side-chains, Ser190 and Gln192; and (3) changes in temperature factors of the pocket walls as well as the introduced P1 side-chain. Binding of the cognate P1 Lys is characterised by almost optimal dihedral angles, hydrogen bonding distances and angles, in addition to considerably lower temperature factors. Thus, the trypsin S1 pocket seems to be designed particularly for lysine binding.     

Conformational behavior of α,α-dialkylated peptides

The preferred conformations of N-acetyl-N′-methyl amides of some dialkylglycines have been determined by empirical conformational-energy calculations; minimum-energy conformations were located by minimizing the energy with respect to all the dihedral angles of the molecules. The conformational space of these compounds is sterically restricted, and low-energy conformations are found only in the regions of fully extended and helical structures. Increasing the bulkiness of the substituents on the C^α, the fully extended conformation becomes gradually more stable than the helical structure preferred in the cases of dimethylglycine. This trend is, however, strongly dependent on the bond angles between the substituents on the C^α atom: In particular, helical structures are favored by standard values (111°) of the N-C^α-C′ angle, while fully extended conformations are favored by smaller values of the same angle, as experimentally observed, for instance, in the case of α,α-di-n-propylglycine.     

Structure and conformation of the disulfide bond in dimeric lung surfactant peptides SP-B1-25 and SP-B8-25

Raman spectroscopy was used to determine the conformation of the disulfide linkage between cysteine residues in the homodimeric construct of the N-terminal alpha helical domain of surfactant protein B (dSP-B(1-25)). The conformation of the disulfide bond between cysteine residues in position 8 of the homodimer of dSP-B(1-25) was compared with that of a truncated homodimer (dSP-B(8-25)) of the peptide having a disulfide linkage at the same position in the alpha helix. Temperature-dependent Raman spectra of the S-S stretching region centered at approximately 500 cm(-1) indicated a stable, although highly strained disulfide conformation with a chi(CS-SC) dihedral angle of +/-10 degrees for the dSP-B(1-25) dimer. In contrast, the truncated dimer dSP-B(8-25) exhibited a series of disulfide conformations with the chi(CS-SC) dihedral angle taking on values of either +/-30 degrees or 85+/-20 degrees . For conformations with chi(CS-SC) close to the +/-90 degrees value, the Raman spectra of the 8-25 truncated dimers exhibited chi(SS-CC) dihedral angles of 90/180 degrees and 20-30 degrees . In the presence of a lipid mixture, both constructs showed a nu(S-S) band at approximately 488 cm(-1), corresponding to a chi(CS-SC) dihedral angle of +/-10 degrees . Polarized infrared spectroscopy was also used to determine the orientation of the helix and beta-sheet portion of both synthetic peptides. These calculations indicated that the helix was oriented primarily in the plane of the surface, at an angle of approximately 60-70 degrees to the surface normal, while the beta structure had approximately 40 degrees tilt. This orientation direction did not change in the presence of a lipid mixture or with temperature. These observations suggest that: (i) the conformational flexibility of the disulfide linkage is dependent on the amino acid residues that flank the cysteine disulfide bond, and (ii) in both constructs, the presence of a lipid matrix locks the disulfide bond into a preferred conformation.     

Structure and dynamics in solution of the complex of Lactobacillus casei dihydrofolate reductase with the new lipophilic antifolate drug trimetrexate

We have determined the three-dimensional solution structure of the complex of Lactobacillus casei dihydrofolate reductase and the anticancer drug trimetrexate. Two thousand seventy distance, 345 dihedral angle, and 144 hydrogen bond restraints were obtained from analysis of multidimensional NMR spectra recorded for complexes containing 15N-labeled protein. Simulated annealing calculations produced a family of 22 structures fully consistent with the constraints. Several intermolecular protein-ligand NOEs were obtained by using a novel approach monitoring temperature effects of NOE signals resulting from dynamic processes in the bound ligand. At low temperature (5 degrees C) the trimethoxy ring of bound trimetrexate is flipping sufficiently slowly to give narrow signals in slow exchange, which give good NOE cross peaks. At higher temperature these broaden and their NOE cross peaks disappear thus allowing the signals in the lower-temperature spectrum to be identified as NOEs involving ligand protons. The binding site for trimetrexate is well defined and this was compared with the binding sites in related complexes formed with methotrexate and trimethoprim. No major conformational differences were detected between the different complexes. The 2,4-diaminopyrimidine-containing moieties in the three drugs bind essentially in the same binding pocket and the remaining parts of their molecules adapt their conformations such that they can make effective van der Waals interactions with essentially the same set of hydrophobic amino acids, the side-chain orientations and local conformations of which are not greatly changed in the different complexes (similar chi1 and chi2 values).     

Structure of the ribotrinucleoside diphosphate codon UpUpC bound to tRNAPhe from yeast. A time-dependent transferred nuclear Overhauser enhancement study

The structure of the ribotrinucleoside diphosphate UpUpC, the codon for phenylalanine, bound to yeast tRNAPhe in solution is elucidated using time-dependent proton-proton transferred nuclear Overhauser enhancement measurements to determine distances between bound ligand protons. The glycosidic bond and ribose conformations are low anti and 3'-endo, respectively, typical of an A-RNA type structure. The main chain torsion angles are all within the range of those expected for A-RNA but small differences from those in conventional A-RNA 11 result in a special structure with a larger rotation per residue (40 to 45 degrees compared to 32.7 degrees in R-RNA 11) and almost perfect stacking of the bases. These two structural features, which are similar to those found in the anticodon triplet of the monoclinic crystal form of tRNAPhe, can account for the known greater stability of the codon-anticodon complex relative to an equivalent double helical RNA trimer with a conventional A-RNA structure.     

Vibrational approach to the dynamics of an α-helix

The dynamics of a finite α-helix have been studied in the harmonic approximation by a vibrational analysis of the atomic motions about their equilibrium positions. The system were represented by an empirical potential energy function, and all degrees of freedom (bond lengths, bond angles, and torsional angles) were allowed to vary. The complete results were compared with a more restrictive model in which the peptide dihedral angle was kept rigid; also, a model potential excluding hydrogen bonds was examined. Thermal fluctuations in the backbone dihedral angles ? and ψ are 12° to 15°. The fluctuations of adjacent dihedral angles are highly correlated, and the correlation pattern is affected by the flexibility of the peptide dihedral angle. Time-dependent autocorrelations in the motion of ? and ψ appear to decay due to dephasing in less than 1 psec, while the motions of the carbonyl oxygen and amide hydrogens out of the peptide plane are more harmonic. Length fluctuations have been evaluated and exhibit a strong end effect; the calculated elastic modulus is in agreement with other values. Rigid and adiabatic total energy surfaces corresponding to dihedral angle rotations in the middle of the helix have been obtained and compared with the quadratic approximation to those surfaces. The magnitudes and correlations between the fluctuations obtained by averaging over the adiabatic energy surface most closely resemble the vibrational results. Of particular interest is the fact that hydrogen bonds play a relatively small role in the local dihedral angle fluctuations, though the hydrogen bonds are important in the energy of overall length changes.     

New strategy for the conformational analysis of carbohydrates based on NOE and 13C NMR coupling constants. Application to the flexible polysaccharide of Streptococcus mitis J22.

For complex oligosaccharides, which are relatively rigid with modest excursions from a single minimum energy conformation, it is straightforward to build conformational models from NOE data. Other oligosaccharides are more flexible with transitions between distinct minima separated by substantial energy barriers. We show that modeling based on scalar coupling data is superior to NOE-based modeling for the latter case. Long range 13C-13C and 13C-1H coupling constants measured for the heptasaccharide repeating subunit of the cell wall polysaccharide from Streptococcus mitis J22 are correlated with individual glycosidic dihedral angles, effectively uncoupling the degrees of freedom of the oligosaccharide and allowing a search for combinations of dihedral angles which are energetically reasonable, i.e., with no bad van der Waals contacts, and which can be combined to satisfy all the measured J values. Allowed values of the individual angles can then be combined to search for overall oligosaccharide conformations which contribute to the ensemble. We show that while the polysaccharide from S. mitis J22 is flexible, requiring multiple conformations, most of the flexibility is localized to a few bonds and only a rather small number of conformations is required to reproduce the experimental NOE and scalar coupling data.     

The sampling properties of some distance geometry algorithms applied to unconstrained polypeptide chains: a study of 1830 independently computed conformations

In this paper we study the statistical geometry of ensembles of poly (L-alanine) conformations computed by several different distance geometry algorithms. Since basic theory only permits us to predict the statistical properties of such ensembles a priori when the distance constraints have a very simple form, the only constraints used for these calculations are those necessary to obtain reasonable bond lengths and angles, together with a lack of short- and long-range atomic overlaps. The geometric properties studied include the squared end-to-end distance and radius of gyration of the computed conformations, in addition to the usual rms coordinate and phi/psi angle deviations among these conformations. The distance geometry algorithms evaluated include several variations of the well-known embed algorithm, together with optimizations of the torsion angles using the ellipsoid and variable target function algorithms. The conclusions may be summarized as follows: First, the distribution with which the trial distances are chosen in most implementations of the embed algorithm is not appropriate when no long-range upper bounds on the distances are present, because it leads to unjustifiably expanded conformations. Second, chosing the trial distances independently of one another leads to a lack of variation in the degree of expansion, which in turn produces a relatively low rms square coordinate difference among the members of the ensemble. Third, when short-range steric constraints are present, torsion angle optimizations that start from conformations obtained by choosing their phi/psi angles randomly with a uniform distribution between -180 degrees and +180 degrees do not converge to conformations whose angles are uniformly distributed over the sterically allowed regions of the phi/psi plane. Finally, in an appendix we show how the sampling obtained with the embed algorithm can be substantially improved upon by the proper application of existing methodology.     

A Karplus equation for (3)J(HCCN) in amino sugar derivatives

The (1)H-(15)N coupling constants of a suite of organic-soluble amino sugar derivatives have been measured by one-dimensional and two-dimensional (1)H/(15)N heteronuclear single quantum, multiple bond correlation (HSQMBC), and the values so obtained are compared with those measured by analysis of (1)H spectra of (15)N-labeled amino sugar derivatives. A number of bicyclic amino sugar models have been studied, including methyl 2- (and 3-)amino-4,6-O-benzylidene-2- (and 3-)deoxy-alpha-D-hexopyranosides in chair or skew conformations, and methyl 2,6-anhydro-3-deoxy-3-phthalimido-alpha-d-mannopyranoside in a locked, almost classical boat conformation. The magnitudes of the vicinal (1)H-(15)N coupling constants (3)J(HCCN) have been correlated with (1)H/(15)N dihedral angles phi computed for the favored conformations by molecular dynamics with molecular mechanics energy minimization. Non-linear regression of the coupling constants on the dihedral angles has yielded a Karplus equation: (3)J(HCCN)=3.1 cos(2) phi-0.6 cos phi+0.4. The coefficients of the terms in this equation have been compared with those reported for 15 other pairs of nuclei, and the coefficient of the important cos(2)phi term found to be numerically smallest for (3)J(HCCN).     

Modeling mutations in protein structures

We describe an automated method for the modeling of point mutations in protein structures. The protein is represented by all non-hydrogen atoms. The scoring function consists of several types of physical potential energy terms and homology-derived restraints. The optimization method implements a combination of conjugate gradient minimization and molecular dynamics with simulated annealing. The testing set consists of 717 pairs of known protein structures differing by a single mutation. Twelve variations of the scoring function were tested in three different environments of the mutated residue. The best-performing protocol optimizes all the atoms of the mutated residue, with respect to a scoring function that includes molecular mechanics energy terms for bond distances, angles, dihedral angles, peptide bond planarity, and non-bonded atomic contacts represented by Lennard-Jones potential, dihedral angle restraints derived from the aligned homologous structure, and a statistical potential for non-bonded atomic interactions extracted from a large set of known protein structures. The current method compares favorably with other tested approaches, especially when predicting long and flexible side-chains. In addition to the thoroughness of the conformational search, sampled degrees of freedom, and the scoring function type, the accuracy of the method was also evaluated as a function of the flexibility of the mutated side-chain, the relative volume change of the mutated residue, and its residue type. The results suggest that further improvement is likely to be achieved by concentrating on the improvement of the scoring function, in addition to or instead of increasing the variety of sampled conformations.