NMR structure refinement

In general, the results of NMR structure refinement should be independent of the applied force field and refinement tools and reflect primarily the experimental restraints. At... 

Iwahara J, Schwieters CD, Clore GM (2004) Ensemble approach for NMR structure refinement against H-1 paramagnetic relaxation enhancement data arising from a flexible paramagnetic group attached to a macromolecule. J Am Chem Soc 126 5879-5896... 

Recent developments in molecular dynamics techniques allow consideration of values and NOE restraints as an ensemble property (Lindorff-Larsen et al. 2005 Richter et al. 2007). The obtained ensembles represent a more realistic view of these flexible molecules in solution than those calculated with conventional NMR structure refinement methods. The dynamically restrained ensembles occupy a considerably larger conformational space than the conventionally calculated ones, and reproduce independent NMR parameters (e.g., chemical shifts) much better. 

A third area where continuum solvent models are useful is in highly constrained simulations. These include X-ray crystallographic and 2D-NMR structural refinements. In these situations, with the additional restraints (and additional computation) arising from the experimental data, the extra expense of explicit solvent models would be inappropriate. 

MD simulations are valuable tools if one wants to gain detailed insight into fast dynamical processes of proteins and other biological macromolecules at atomic resolution. But since conventional MD simulations are confined to the study of very fast processes, conformational flooding represents a complementary and powerful tool to predict and understand also slow conformational motions. Another obvious application is an enhanced refinement of Xray- or NMR-structures. 

The greatest value of molecular dynamic simulations is that they complement and help to explain existing data for designing new experiments. The simulations are increasingly useful for structural refinement of models generated from NMR, distance geometry, and X-ray data. 

The first step for any structure elucidation is the assignment of the frequencies (chemical shifts) of the protons and other NMR-active nuclei ( C, N). Although the frequencies of the nuclei in the magnetic field depend on the local electronic environment produced by the three-dimensional structure, a direct correlation to structure is very complicated. The application of chemical shift in structure calculation has been limited to final structure refinements, using empirical relations [14,15] for proton and chemical shifts and ab initio calculation for chemical shifts of certain residues [16]. 

The well-known difficulties in calculating tliree-dimensional structures of macromolecules from NMR data mentioned above (sparseness of the data, imprecision of the restraints due to spin diffusion and internal dynamics) also make the validation of the structures a challenging task. The quality of the data [88] and the energy parameters used in the refinement [89] can be expected to influence the quality of structures. Several principles can be used to validate NMR structures. 

First, the structure should explain the data. Apart from the energy or target function value returned by the refinement program, this check can be performed with some independent programs (e.g., AQUA/PROCHECK-NMR [90], MOLMOL [91]). The analysis of the deviations from the restraints used in calculating the structures is very useful in the process of assigning the NOE peaks and refining the restraint list. As indicators of the quality of the final structure they are less powerful, because violations have been checked and probably removed. A recent statistical survey of the quality of NMR structures found weak correlations between deviations from NMR restraints and other indicators of structure quality [88]. 

D Case. New directions m NMR spectral simulation and structure refinement. In WF van Gunsteren, PK Weiner, AJ WiUcmson, eds. Computer Simulation of Biomolecular Systems Theoretical and Experimental Applications, Vol 2. Leiden ESCOM, 1993, pp 382-406. 

To put the errors in comparative models into perspective, we list the differences among strucmres of the same protein that have been detennined experimentally (Fig. 9). The 1 A accuracy of main chain atom positions corresponds to X-ray structures defined at a low resolution of about 2.5 A and with an / -factor of about 25% [192], as well as to medium resolution NMR structures determined from 10 interproton distance restraints per residue [193]. Similarly, differences between the highly refined X-ray and NMR structures of the same protein also tend to be about 1 A [193]. Changes in the environment... 

More detailed aspects of protein function can be obtained also by force-field based approaches. Whereas protein function requires protein dynamics, no experimental technique can observe it directly on an atomic scale, and motions have to be simulated by molecular dynamics (MD) simulations. Also free energy differences (e.g. between binding energies of different protein ligands) can be characterised by MD simulations. Molecular mechanics or molecular dynamics based approaches are also necessary for homology modelling and for structure refinement in X-ray crystallography and NMR structure determination. 

Pintacuda, G., Moshref, A., Leonchiks, A., Sharipo, A., Otting, G. Site-specific labeling with a metal chelator for protein-structure refinement. /. Biomol. NMR 2004, 29, 351-361. 

Torda, A. E., Brunne, R. M., Huber, T., Kessler, H., Van Gunsteren, W. F. Structure refinement using time-averaged /-coupling constant resttaints. /. Biomol. NMR 1993, 3, 55-66. 

Other exciting applications involved using parallel tempering in connection with available experimental data. For example, Falcioni and Deem [57] used X-ray data to refine structures of zeolites, and Haliloglu et al. [58] refined NMR structural data for proteins (in particular using residual dipolar coupling constraints). 

The CSA is sensitive to structural properties, and they may be used for structure refinement based on NMR data, as will be further discussed in Sect. 2.2.2. 

For molecular sizes that are amenable by NMR techniques, nucleic acids usually lack a tertiary fold. This fact, together with the characteristic low proton density, complicates NMR structural analysis of nucleic acids. As a result, local geometries and overall shapes of nucleic acids, whose structures have been determined by NMR, usually are poorly defined. Dipolar couplings provide the necessary long-range information to improve the quality of nucleic acid structures substantially [72]. Some examples can be found already in the literature where the successful application of dipolar couplings into structure calculation and structure refinement of DNA and RNA are reported [73-77]. 

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