Distance restraints structure refinement

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... 

The first step of the structure refinement is the appHcation of distance geometry (DG) calculations which do not use an energy function but only experimentally derived distances and restraints which follow directly from the constitution, the so-caUed holonomic constraints. Those constraints are, for example, distances between geminal protons, which normally are in the range between 1.7 and 1.8 A, or the distance between vicinal protons, which can not exceed 3.1 A when protons are in anti-periplanar orientation. 

This method of refinement was first applied, using the distance restraint form of Eq. [9], to the lac repressor headpiece using a model as the initial structure.9-83 It was later applied to a heptadecapeptide.84 Subsequent studies with model data showed that, with a carefully chosen simulation protocol, the method was capable of refining even poor starting structures.85 86... 

Since this early work, experience has accumulated with restrained MD and some points can be made with respect to the effects of restrained MD and simulation protocol. First, if the starting structures have already been very highly refined with respect to distances, and if the distance restraints are very numerous and accurate, then the restrained MD protocol will not make much difference. If the starting structures are less well refined or the restraints less numerous, then restrained MD will be important, as will the choice of protocol. 

When making such a decision, some considerations should be borne in mind. First, if one is using a refinement scheme that produces a known distribution of structures, then one can calculate the likely deviation that the pseudo-energy terms will permit. For example, if an MD refinement is used, structures will be able to cross barriers of about BT. Then, if one is using a quadratic form to enforce distance restraints, one could recast Eq. [9] as follows to get an idea of the violations that would be permitted for a given force constant and temperature ... 

This style of refinement also reflected a change of emphasis with respect to the computational balance between initial structure generation and restrained MD. Previously, most workers had spent much time refining structures with respect to distance restraints before applying MD. With this modified force field, it was practical to start MD refinement with crude metric matrix structures after only substructure embedding, or even simply from extended strand initial structures. 

One important application of RDC measurements is the structural refinement of biomolecules that consist of several domains that are connected by more or less flexible linkers. Because of the flexibility of the linker and the distance between domains, J-couplings and NOE restraints will frequently not be sufficient for correct determination of the relative domain orientation. The addition of RDC restraints in structure calculation not only refines the biomolecular structure but also allows the relationship between structure and function to be studied. Interactions with other biomolecules and ligand binding may induce an intramolecular rearrangement of the relative orientation of domains that is detectable through RDC measurements (21, 22). 

Generally, twinned crystals tend to have a poor effective data to parameter ratio, so they often require restraints in order to obtain a satisfactory refinement (Watkin, 1994). The following restraints can be useful distance restraints for chemically equivalent 1,2- and 1,3-distances, planarity restraints for groups such as phenyl rings, rigid bond ADP restraints (Hirshfeld, 1976 Rollett, 1970 Traeblood and Dunitz, 1983) and similar ADP restraints (Sheldrick, 1997b). Even when restraints are employed, the distribution of the displacement parameters (ORTEPplot) and residual features in a difference electron density map can be less satisfactory than for a normal structure determination. 

These effects are large enough to make it mandatory to correct NOE intensities before taking the ensuing complete relaxation matrix-derived distance restraints into structure refinement calculations. Naturally, this step is less important when NOE intensities are... 

The preliminary NMR structure of SRP 28mer is shown in Figure 8. We are currently performing the final rMD refinement simulations with improved MARDIGRAS-derived distance restraints where the preliminary NMR structure was used as a starting model for the MARDIGRAS calculations. Therefore, we refrain from a detailed discussion of the structure of the SRP 28mer in this article and refer to future publications. 

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