Spanish National Microbiology Centre, Institute of Health Carlos III, Madrid, Spain.
Department of Structure of Macromolecules, Centro Nacional de Biotecnología (CNB-CSIC), Campus de Cantoblanco, Madrid, Spain.
J Mol Biol. 2023 Apr 15;435(8):168024. doi: 10.1016/j.jmb.2023.168024. Epub 2023 Feb 23.
The biological function of macromolecular complexes depends not only on large-scale transitions between conformations, but also on small-scale conformational fluctuations at equilibrium. Information on the equilibrium dynamics of biomolecular complexes could, in principle, be obtained from local resolution (LR) data in cryo-electron microscopy (cryo-EM) maps. However, this possibility had not been validated by comparing, for a same biomolecular complex, LR data with quantitative information on equilibrium dynamics obtained by an established solution technique. In this study we determined the cryo-EM structure of the minute virus of mice (MVM) capsid as a model biomolecular complex. The LR values obtained correlated with crystallographic B factors and with hydrogen/deuterium exchange (HDX) rates obtained by mass spectrometry (HDX-MS), a gold standard for determining equilibrium dynamics in solution. This result validated a LR-based cryo-EM approach to investigate, with high spatial resolution, the equilibrium dynamics of biomolecular complexes. As an application of this approach, we determined the cryo-EM structure of two mutant MVM capsids and compared their equilibrium dynamics with that of the wild-type MVM capsid. The results supported a previously suggested linkage between mechanical stiffening and impaired equilibrium dynamics of a virus particle. Cryo-EM is emerging as a powerful approach for simultaneously acquiring information on the atomic structure and local equilibrium dynamics of biomolecular complexes.
大分子复合物的生物学功能不仅取决于构象之间的大规模转变，还取决于平衡时的小规模构象波动。原则上，可以从冷冻电子显微镜（cryo-EM）图谱的局部分辨率（LR）数据中获取生物分子复合物的平衡动力学信息。然而，这种可能性尚未通过将同一生物分子复合物的 LR 数据与通过成熟的溶液技术获得的关于平衡动力学的定量信息进行比较来验证。在这项研究中，我们确定了微小病毒（MVM）衣壳作为模型生物分子复合物的冷冻电镜结构。获得的 LR 值与晶体学 B 因子相关，并且与通过质谱法（HDX-MS）获得的氢/氘交换（HDX）速率相关，HDX-MS 是确定溶液中平衡动力学的金标准。这一结果验证了一种基于 LR 的冷冻电镜方法，该方法可用于以高空间分辨率研究生物分子复合物的平衡动力学。作为该方法的应用，我们确定了两种突变 MVM 衣壳的冷冻电镜结构，并将它们的平衡动力学与野生型 MVM 衣壳进行了比较。结果支持了先前提出的病毒颗粒机械硬度增强与平衡动力学受损之间的联系。冷冻电镜正在成为一种强大的方法，可以同时获取生物分子复合物的原子结构和局部平衡动力学信息。
