In structure determination by X-ray crystallography and solution NMR
spectroscopy, experimental data are collected as time- and ensemble-averages.
Thus, in principle, appropriate time- and ensemble-averaged models should be
used. Refinement of an ensemble of conformers rather
than one single structure against the experimental NMR data could, however,
result in overfitting the data because of the significantly increased number of
parameters. To avoid overfitting, complete cross-validation (Bruenger, 1992; Bruenger et al., 1993), which provides an
unbiased measure of the fit, has been applied to NOE derived distance
refinement. Using two synthetic test cases ( protein G and AmbtV), a correlation was demonstrated
between the cross-validated measure of the fit (defined in terms of rms
deviations from the distance restraints and number of violations) and the
number of models that best reproduce the conformational variability in
solution. A new method, based on a probability map, has been used to generate
good representations of the resulting ensembles of structures. The method has
also been applied to observed NMR data for two proteins, interleukin 4 and
interleukin 8 (Clore et al., 1990). For interleukin 4, cross-validation indicates that a
single-conformer model gives the most accurate representation of the structure
whereas conventional measures of fit between the experimental data and those
calculated from the model decrease when increasing the number of conformers,
indicating overfitting. For interleukin 8, complete cross-validation predicts a
twin-conformer model to be the most faithful representation of the experimental
data. Two distinct conformations for the loop formed by residues 16 to 22
emerge from the family of twin-conformer structures. The putative alternate
conformation of the loop is not observed in the crystal structure of
interleukin 8 (Baldwin et al., 1991). However, because of crystal packing contacts in this region this
does not necessarily exclude the presence of the alternate conformation in
solution. The twin-conformer model is supported by
observed chemical exchange line broadening for the amide of His-18 obtained by
15N relaxation studies (Grasberger et al., 1993). This region has also been implied to be involved in
receptor binding (Clubb et al.,1994).
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