Interdisciplinary Research Center, HALOmem, Martin Luther University of Halle-Wittenberg, Kurt-Mothes-Straße 3, 06120 Halle, Germany.
Bioanalytical Mass Spectrometry Research Group, Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, 37077 Göttingen, Germany; Bioanalytics, Department of Clinical Chemistry, University Medical Center Göttingen, Robert-Koch-Straße 40, 37075 Göttingen, Germany.
Curr Opin Struct Biol. 2017 Oct;46:157-168. doi: 10.1016/j.sbi.2017.10.005. Epub 2017 Nov 5.
Determining the structures of, and gaining insight into, the function of large protein complexes at the molecular or atomic level has become a key part of modern structural biology. Electron cryo-microscopy (cryo-EM) can solve structures of highly dynamic macromolecular complexes that are not feasible with other structural techniques like X-ray of crystallized proteins (protein complexes) or nuclear magnetic resonance (NMR) spectroscopy of proteins (protein complexes) in solution. To resolve the regions that are less well defined in cryo-EM images, cross-linking coupled with mass spectrometry (CX-MS) provides valuable information on the proximity between amino-acid residues as distance constraints for homology or de novo modelling. The CX-MS strategy involves covalent linkage, with chemical cross-linkers, of residues close to each other in three-dimensional space and identifying these connections by mass spectrometry. In this article, we summarise the advances of CX-MS and its integration with cryo-EM for structural reconstruction. We further evaluate a number of important examples of structure determination that followed this combinatorial strategy.
确定大型蛋白质复合物在分子或原子水平上的结构，并深入了解其功能，已成为现代结构生物学的重要组成部分。电子冷冻显微镜（cryo-EM）可解决其他结构技术（如 X 射线结晶蛋白（蛋白质复合物）或溶液中蛋白质的核磁共振（NMR）光谱学）无法解决的高度动态大分子复合物的结构。为了解决冷冻电镜图像中定义不明确的区域，交联与质谱（CX-MS）相结合，为同源建模或从头建模提供了有关氨基酸残基之间接近程度的有价值的信息，作为距离约束。CX-MS 策略涉及用化学交联剂将三维空间中彼此靠近的残基共价连接，并通过质谱鉴定这些连接。在本文中，我们总结了 CX-MS 的进展及其与冷冻电镜在结构重建方面的结合。我们进一步评估了一些遵循这种组合策略的重要结构测定的例子。
