Multi-crystal native-SAD phasing at 5 keV with a helium environment.
Multi-crystal native-SAD phasing at 5 keV with a helium environment.
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使用氦路径的低能量天然 SAD 定相可以更常规地用于解决具有挑战性的膜蛋白结构。 使用生物分子中的天然硫或磷（天然-SAD）通过单波长异常衍射进行从头结构测定是一种颇具吸引力的方法，可减少重原子衍生物和硒代甲硫氨酰取代的劳动密集型生产。天然 SAD 方法对于膜蛋白特别有吸引力，因为膜蛋白难以生产并且通常难以生长成适当大小的晶体。 Native-SAD 使用较低能量的 X 射线来增强硫或磷的异常信号。然而，在较低能量下，空气的散射和吸收会增加背景噪声，减少信号，从而不利于本地 SAD 定相。我们之前已经在 NSLS-II FMX 光束线中在空气中以 5 keV 的能量演示了原生 SAD 定相。这里描述了使用氦气路径来降低背景散射噪声和衍射 X 射线束的空气吸收。氦气路径用于收集两种蛋白质在 5 keV 下的异常衍射数据：索马甜和膜蛋白 TehA。尽管来自每个单独晶体的异常信号非常微弱，但从微米大小晶体组装的数据中可以获得强大的异常信号。从 15 个微晶中确定了索马甜结构，并从 18 个微晶中确定了 TehA 结构。这些结果证明了氦环境在支持 5 keV 的天然 SAD 定相方面的有用性。
Low-energy native-SAD phasing using a helium path could be used more routinely for solving challenging membrane protein structures. De novo structure determination from single-wavelength anomalous diffraction using native sulfur or phosphorus in biomolecules (native-SAD) is an appealing method to mitigate the labor-intensive production of heavy-atom derivatives and selenomethionyl substitutions. The native-SAD method is particularly attractive for membrane proteins, which are difficult to produce and often recalcitrant to grow into decent-sized crystals. Native-SAD uses lower-energy X-rays to enhance anomalous signals from sulfur or phosphorus. However, at lower energies, the scattering and absorption of air contribute to the background noise, reduce the signals and are thus adverse to native-SAD phasing. We have previously demonstrated native-SAD phasing at an energy of 5 keV in air at the NSLS-II FMX beamline. Here, the use of a helium path developed to reduce both the noise from background scattering and the air absorption of the diffracted X-ray beam are described. The helium path was used for collection of anomalous diffraction data at 5 keV for two proteins: thaumatin and the membrane protein TehA. Although anomalous signals from each individual crystal are very weak, robust anomalous signals are obtained from data assembled from micrometre-sized crystals. The thaumatin structure was determined from 15 microcrystals and the TehA structure from 18 microcrystals. These results demonstrate the usefulness of a helium environment in support of native-SAD phasing at 5 keV.