Department of Molecular and Cellular Biology, Harvard University, 52 Oxford Street, Cambridge, Massachusetts, USA.
Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts, USA.
Acta Crystallogr D Struct Biol. 2022 Aug 1;78(Pt 8):986-996. doi: 10.1107/S2059798322006799. Epub 2022 Jul 27.
Single-wavelength anomalous diffraction (SAD) is a routine method for overcoming the phase problem when solving macromolecular structures. This technique requires the accurate measurement of intensities to determine differences between Bijvoet pairs. Although SAD experiments are commonly conducted at cryogenic temperatures to mitigate the effects of radiation damage, such temperatures can alter the conformational ensemble of the protein and may impede the merging of data from multiple crystals due to non-uniform freezing. Here, a strategy is presented to obtain high-quality data from room-temperature, single-crystal experiments. To illustrate the strengths of this approach, native SAD phasing at 6.55 keV was used to solve four structures of three model systems at 295 K. The resulting data sets allow automatic phasing and model building, and reveal alternate conformations that reflect the structure of proteins at room temperature.
单波长反常散射（SAD）是解决大分子结构相位问题的常规方法。该技术需要精确测量强度以确定对映体间的差异。尽管 SAD 实验通常在低温下进行以减轻辐射损伤的影响，但这种温度会改变蛋白质的构象整体，并且可能由于不均匀冻结而阻碍来自多个晶体的数据的合并。这里提出了一种在室温下进行单晶实验的方法，以获得高质量的数据。为了说明该方法的优势，使用 6.55keV 的原生 SAD 相位法在 295K 下解决了三个模型系统的四个结构。所得数据集允许自动相和模型构建，并揭示了反映蛋白质在室温下结构的替代构象。
