Structure determination restraints

Figure 3 Model building by Modeller [31], First, spatial restraints in the form of atomic distances and dihedral angles are extracted from the template stmcture(s). The alignment is used to determine equivalent residues between the target and the template. The restraints are combined into an objective function. Finally, the model for the target is optimized until a model that best satisfies the spatial restraints is obtained. This procedure is technically similar to the one used in structure determination by NMR.

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... 

CM Clore, AT Bifinger, M Karplus, AM Gronenborn. Application of molecular dynamics with mterproton distance restraints to 3D protein structure determination. J Mol Biol 191 523-551, 1986. 

Fig. 9.4 The fixation of the Ala proton in cyclosporin A via ROE-derived distance restraints as an illustrative example for NMR structure determination. (A) The amide region of the 2D ROESY spectrum of cyclosporin A at room temperature with the cross-peaks to...

In the case of being successful in calculating multiple conformations by using time- or ensemble-averaged MD restraints the solved molecular structures are presented as 3D models and can be deposited in an electronic structure database (17). Finally, it is recommended to provide an accurate explanation of the procedures used for the structure elucidation because the application of different methods (NMR, DG, MD, SA, Monte-Carlo calculations. X-ray crystallography) may result in varying conformational models which do not implicitly display the real state of a molecule. This aspect should be always kept in mind when dealing with structure determination methods. 

The lack of long-range restraints has hindered the precise and accurate structure determination of nucleic acids for a long time. The isotopic enrichment mostly improved the accuracy of local geometry without a pronounced influence on the overall shape. However,... 

Dipolar couplings are very powerful restraints for structure determination of biomolecules and the determination of protein-protein or protein-ligand interactions [16]. They depend on the orientation of an internuclear vector, and its distance and the angular dependence is given by the following formula ... 

As another example, the three-dimensional structure of Cytochrome c has been determined on the basis of structural information from pseudocontact paramagnetic chemical shifts, Curie-Dipolar cross-correlation, secondary structure constraints, dipolar couplings and 15N relaxation data [103]. This protein has a paramagnetic center, and therefore the above-mentioned conformational restraints can be derived from this feature. Dipolar couplings do not average to zero because of the susceptibility tensor anisotropy of the protein. The structure determination of this protein without NOE data gives an RMSD (root... 

The solution structures of 1 1 complex between paromomycin and gentamicin Cla and the A-site oligonucleotide have been solved at high resolution using heteronuclear NMR techniques These approaches require the preparation of uniformly labeled N- and C-RNA oligonucleotides via in vitro transcription with labeled nucleoside triphosphates. The use of RNAs labeled with NMR-active nuclei allows for the application of sophisticated heteronuclear NMR methods. These methods facilitate the assigmnent of NMR resonances and the acquisition of structural restraints for detailed structure determinations. 

There are six subunits in the asymmetric unit of the Panuliris structure, arranged in a particle best described as a trimer of dimers. The 3-2 symmetry was good enough to be useful in a phase extension technique commonly used in the structure determination of highly symmetric viruses. Unfortunately, these crystals diffract only to 3.2 A, so that there cannot be a high level of detail available for description of the active site. Nevertheless, because there are six independent copies of the molecule in the asymmetric unit, there is more information than might normally be available at this resolution. The structure has been carefully refined at this resolution, with and without the 3-2 symmetry restraint. The estimated coordinate error is —0.35 A. The overall R is 0.201 for data between 8 and 3.2 A. Surprisingly, while the overalls for subunits 1 and... 

In this section, I provide a simplified physical picture of pulse NMW spectroscopy, including a simple conceptual model to help you understand multidimensional NMR. Then I briefly discuss the problems of assigning resonances and determining distance restraints for molecules as large and complex as proteins, and the methods for deriving a structure from this information. Finally, I discuss the contents of coordinate files from NMR structure determination and provide some hints on judging the quality of models. 

At first glance, the averaged model would appear to serve most researchers who are looking for a molecular model to help them explain the function of the molecule and rationalize other chemical, spectroscopic, thermodynamic, and kinetic data. On the other hand, you might think that the ensemble and distance-restraint files are of most use to those working to improve structure determination techniques. There are good reasons however, for all researchers to look carefully at the ensemble, as discussed in the next section. 

In a typical free NOESY experiment of a molecule in the absence of any interacting protein, cross-peak volumes are interpreted in terms of a set of interproton distances r that can be used as distance restraints in structure determination procedures, like restrained simulated annealing protocols [44], In a tr-NOESY, i.e. a NOESY measured under exchange-transferred conditions in the presence of a protein - i.e., an excess of soluble ligand is in fast exchange equilibrium with a smaller amount of protein-bound ligand -, these r reflect the interproton distances of the ligand in the bound... 

The main source of conformational information for biopolymers are the easy-to-obtain chemical shifts that can be translated into dihedral restraints. In addition, for fully 13C labeled compounds, proton-driven spin diffusion between carbons [72] can be used to measure quantitatively distances between carbons. The CHHC experiment is the equivalent of the NOESY in solution that measures distances between protons by detecting the resonances of the attached carbons. While both techniques, proton-driven spin diffusion and CHHC experiment [73], allow for some variation in the distance as determined from cross-peak integrals, REDOR [74] experiments in selective labeled compounds measure very accurate distances by direct observation of the oscillation of a signal by the dipolar coupling. While the latter technique provides very accurate distances, it provides only one piece of information per sample. Therefore, the more powerful techniques proton-driven spin diffusion and CHHC have taken over when it comes to structure determination by ss-NMR of fully labeled ligands. 

The structure determination of biopolymers using NMR spectroscopy usually involves interactions of protons[216,33. Typically, interactions of protons (nuclear Overhauser effect, NOE) that are close in space but separated by several subunits of the biopolymer are used to establish the folding of the backbone. Distance restraints are then used to compute a structure which is checked by back-calculation of the NOE spectra and comparison with experimental results 361. For large and highly flexible systems molecular dynamics is invaluable for scanning the conformational space. 

There are three aspects to consider. First, we summarize briefly the underlying computational framework needed and the general strategy used in the structure determination. Second, we cover the use of 2D, 3D, and 4D methods to permit the sequential assignment of peaks to specific amino acids. Finally, we describe the use of nuclear Overhauser enhancements and spin coupling constants to provide restraints on interproton distances and bond angles, and we indicate how dipolar coupling and chemical shifts can sometimes add further information on molecular conformation. 

Potluri S, Yan AK, Chou JJ, Donald BR, Bailey-Kellogg C. Structure determination of symmetric homo-oligomers by a complete search of symmetry configuration space, using NMR restraints and van der Waals packing. Proteins 2006 65 203-219. 

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