Department of Physics, Nanosystems Initiative Munich, and Center for Nanoscience, LMU Munich, Munich, Germany.
Department of Biochemistry, Stanford University, Stanford, CA 94305, USA.
Sci Adv. 2018 May 25;4(5):eaar4418. doi: 10.1126/sciadv.aar4418. eCollection 2018 May.
Small-angle x-ray scattering (SAXS) is a powerful technique to probe the structure of biological macromolecules and their complexes under virtually arbitrary solution conditions, without the need for crystallization. While it is possible to reconstruct molecular shapes from SAXS data ab initio, the resulting electron density maps have a resolution of 1 nm and are often insufficient to reliably assign secondary structure elements or domains. We show that SAXS data of gold-labeled samples significantly enhance the information content of SAXS measurements, allowing the unambiguous assignment of macromolecular sequence motifs to specific locations within a SAXS structure. We first demonstrate our approach for site-specifically internally and end-labeled DNA and an RNA motif. In addition, we present a protocol for highly uniform and site-specific labeling of proteins with small (1.4 nm diameter) gold particles and apply our method to the signaling protein calmodulin. In all cases, the position of the small gold probes can be reliably identified in low-resolution electron density maps. Enhancing low-resolution measurements by site-selective gold labeling provides an attractive approach to aid modeling of a large range of macromolecular systems.
小角 X 射线散射（SAXS）是一种强大的技术，可以在几乎任意的溶液条件下探测生物大分子及其复合物的结构，而无需结晶。虽然可以从头开始从 SAXS 数据重建分子形状，但得到的电子密度图的分辨率约为 1nm，并且通常不足以可靠地分配二级结构元件或结构域。我们表明，金标记样品的 SAXS 数据显著增加了 SAXS 测量的信息量，允许将大分子序列基序明确分配给 SAXS 结构内的特定位置。我们首先证明了我们的方法可用于特异性内部和末端标记的 DNA 和 RNA 基序。此外，我们还提出了一种用于用小（约 1.4nm 直径）金颗粒高度均匀和特异性标记蛋白质的方案，并将我们的方法应用于信号蛋白钙调蛋白。在所有情况下，小的金探针的位置都可以在低分辨率电子密度图中可靠地识别。通过选择性金标记增强低分辨率测量为帮助建模提供了一种有吸引力的方法，适用于各种大分子系统。
