Robust structural analysis of native biological macromolecules from multi-crystal anomalous diffraction data.
Robust structural analysis of native biological macromolecules from multi-crystal anomalous diffraction data.
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DOI：
10.1107/s0907444913001479
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发表时间：
2013-07
期刊：
影响因子：
--
通讯作者：
Hendrickson WA
中科院分区：
文献类型：
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作者：
Liu Q;Liu Q;Hendrickson WA
来自典型天然大分子的异常衍射信号非常弱，阻碍了它们在结构测定中的使用。这里，描述了通过使用多个晶体来增强反常衍射中的信噪比的本机 SAD 程序。五项应用表明，无需重原子即可实现真正的常规结构测定。没有已知结构前因的生物大分子的结构测定通常涉及掺入比生物分子中天然存在的原子更重的原子。目前，使用单波长或多波长异常衍射（SAD 或 MAD）数据分析硒代甲硫氨酰蛋白在此类从头分析中占主导地位。天然存在的金属离子（例如锌或铁）通常足以满足 MAD 或 SAD 实验的要求，而硫 SAD 自从 30 年前首次使用 Crambin 进行演示以来一直是一种选择；然而，仅包含轻原子（Z max ≤ 20）的结构的 SAD 分析并不常见。在此，描述了通过组合以低于通常的 X 射线能量从多个晶体收集的数据来增强异常衍射测量中的信噪比的稳健程序。这种多晶天然 SAD 方法应用于五种结构测定，使用 5 到 13 个晶体来测定 4 到 52 个异常散射体 (Z ≤ 20) 的子结构，然后以 2.3 到 2.8 Å 的分辨率确定每个不对称单元 127 到 1200 个有序残基的完整结构。设计测试以确保所使用的所有晶体在统计上是等效的。 Ca、Cl、S、P 和 Mg 的元素身份通过 f'' 散射因子细化得到证明。该程序是稳健的，表明实现了典型天然大分子的真正常规结构测定。针对低能 X 射线衍射测量进行优化的同步加速器光束线将有助于这种直接结构分析。
Anomalous diffraction signals from typical native macromolecules are very weak, frustrating their use in structure determination. Here, native SAD procedures are described for enhancing the signal to noise in anomalous diffraction by using multiple crystals are described. Five applications demonstrate that truly routine structure determination is possible without the need for heavy atoms. Structure determinations for biological macromolecules that have no known structural antecedents typically involve the incorporation of heavier atoms than those found natively in biological molecules. Currently, selenomethionyl proteins analyzed using single- or multi-wavelength anomalous diffraction (SAD or MAD) data predominate for such de novo analyses. Naturally occurring metal ions such as zinc or iron often suffice in MAD or SAD experiments, and sulfur SAD has been an option since it was first demonstrated using crambin 30 years ago; however, SAD analyses of structures containing only light atoms (Z max ≤ 20) have not been common. Here, robust procedures for enhancing the signal to noise in measurements of anomalous diffraction by combining data collected from several crystals at a lower than usual X-ray energy are described. This multi-crystal native SAD method was applied in five structure determinations, using between five and 13 crystals to determine substructures of between four and 52 anomalous scatterers (Z ≤ 20) and then the full structures ranging from 127 to 1200 ordered residues per asymmetric unit at resolutions from 2.3 to 2.8 Å. Tests were devised to assure that all of the crystals used were statistically equivalent. Elemental identities for Ca, Cl, S, P and Mg were proven by f′′ scattering-factor refinements. The procedures are robust, indicating that truly routine structure determination of typical native macromolecules is realised. Synchrotron beamlines that are optimized for low-energy X-ray diffraction measurements will facilitate such direct structural analysis.