Circular dichroism (CD) spectroscopy is highly sensitive to the secondary structure (SS) composition of proteins. Several methods exist to either estimate the SS composition of a protein or to validate existing structural models using its CD spectrum. The accuracy and precision of these methods depend on the quality of both the measured CD spectrum and the used reference structure. Using a large reference protein set with high-quality CD spectra and synthetic data derived from this set, we quantified deviations from both ideal spectra and reference structures due to experimental limitations. We also determined the impact of these deviations on SS estimation, CD prediction, and SS validation methods of the SESCA analysis package. With regard to the CD spectra, our results suggest intensity scaling errors and non-SS contributions as the main causes of inaccuracies. These factors also can lead to overestimated model errors during validation. The errors of the used reference structures combine non-additively with errors caused by the CD spectrum, which increases the uncertainty of model validation. We have further shown that the effects of scaling errors in the CD spectrum can be nearly eliminated by appropriate re-scaling, and that the accuracy of model validation methods can be improved by accounting for typical non-SS contributions. These improvements have now been implemented within the SESCA package and are available at: https://www.mpibpc.mpg.de/sesca.

圆二色性（CD）光谱对蛋白质的二级结构（SS）组成高度敏感。存在几种估计蛋白质的SS组成或使用其CD光谱验证现有结构模型的方法。这些方法的准确性和精度取决于所测CD光谱和所用参考结构的质量。使用具有高质量CD光谱的大型参考蛋白质组和从该组衍生的合成数据，由于实验的局限性，我们量化了与理想光谱和参考结构的偏差。我们还确定了这些偏差对SESCA分析软件包的SS估计，CD预测和SS验证方法的影响。关于CD光谱，我们的结果表明强度缩放比例误差和非SS贡献是导致误差的主要原因。这些因素也可能导致验证期间高估模型误差。所用参考结构的误差与CD谱图引起的误差相加地相加，从而增加了模型验证的不确定性。我们进一步表明，通过适当的重新缩放，可以几乎消除CD频谱中缩放误差的影响，并且可以通过考虑典型的非SS贡献来提高模型验证方法的准确性。这些改进现已在SESCA软件包中实现，并且可以从以下网址获得：https://www.mpibpc.mpg.de/sesca。所用参考结构的误差与CD谱图引起的误差相加地相加，从而增加了模型验证的不确定性。我们进一步表明，通过适当的重新缩放，可以几乎消除CD频谱中缩放误差的影响，并且可以通过考虑典型的非SS贡献来提高模型验证方法的准确性。这些改进现已在SESCA软件包中实现，并且可以从以下网址获得：https://www.mpibpc.mpg.de/sesca。所用参考结构的误差与CD谱图引起的误差相加地相加，从而增加了模型验证的不确定性。我们进一步表明，通过适当的重新缩放，可以几乎消除CD频谱中缩放误差的影响，并且可以通过考虑典型的非SS贡献来提高模型验证方法的准确性。这些改进现已在SESCA软件包中实现，并且可以从以下网址获得：https://www.mpibpc.mpg.de/sesca。并且通过考虑典型的非SS贡献可以提高模型验证方法的准确性。这些改进现已在SESCA软件包中实现，并且可以从以下网址获得：https://www.mpibpc.mpg.de/sesca。并且通过考虑典型的非SS贡献可以提高模型验证方法的准确性。这些改进现已在SESCA软件包中实现，并且可以从以下网址获得：https://www.mpibpc.mpg.de/sesca。