Protonation-state determination in proteins using high-resolution X-ray crystallography: effects of resolution and completeness
Protonation-state determination in proteins using high-resolution X-ray crystallography: effects of resolution and completeness
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DOI：
10.1107/s0907444912012589
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发表时间：
2012-07-01
影响因子：
2.2
通讯作者：
Helliwell, J. R.
中科院分区：
文献类型：
--
作者：
Fisher, S. J.;Blakeley, M. P.;Helliwell, J. R.
用键距分析确定了胰蛋白酶、枯草菌素和溶菌酶中可电离氨基酸的质子化状态。胰蛋白酶的衍射分辨率为1.2埃（97%完成，2.5 sigma时h原子可见度为12%），枯草菌素的衍射分辨率为1.26埃（100%完成，2.5 sigma时h原子可见度为11%），溶菌酶的衍射分辨率为0.65埃（PDB入口2vb1； 98%完成，3 sigma时h原子可见度为30%）。这些研究为评估提供了广泛的衍射分辨率范围。所获得的键长esds小至0.008埃，因此为键长分析提供了绝佳的机会。结果表明，在1.2 ~ 1.3埃的分辨率下，衍射数据可以获得有用的信息，并且分辨率的微小增加可以显著降低相关的键长标准偏差。还考虑了组氨酸残基中的质子化状态；然而，由于质子化和去质子化形式之间的差异较小，因此推断这些残基的质子化状态要困难得多。即使是0.65埃分辨率的溶菌酶结构也不能提供必要的准确性来确定组氨酸的质子化状态。
A bond-distance analysis has been undertaken to determine the protonation states of ionizable amino acids in trypsin, subtilisin and lysozyme. The diffraction resolutions were 1.2 angstrom for trypsin (97% complete, 12% H-atom visibility at 2.5 sigma), 1.26 angstrom for subtilisin (100% complete, 11% H-atom visibility at 2.5 sigma) and 0.65 angstrom for lysozyme (PDB entry 2vb1; 98% complete, 30% H-atom visibility at 3 sigma). These studies provide a wide diffraction resolution range for assessment. The bond-length e.s.d.s obtained are as small as 0.008 angstrom and thus provide an exceptional opportunity for bond-length analyses. The results indicate that useful information can be obtained from diffraction data at around 1.2-1.3 angstrom resolution and that minor increases in resolution can have significant effects on reducing the associated bond-length standard deviations. The protonation states in histidine residues were also considered; however, owing to the smaller differences between the protonated and deprotonated forms it is much more difficult to infer the protonation states of these residues. Not even the 0.65 angstrom resolution lysozyme structure provided the necessary accuracy to determine the protonation states of histidine.