Abstract：
Single particle analysis from cryogenic transmission electron microscopy (cryo-EM) is particularly attractive for complexes for which structure prediction remains intractable, such as antibody-antigen complexes. Here we obtain the detailed structure of a particularly difficult complex between human epidermal growth factor receptor 2 (HER2) and the antigen-binding fragments from two distinct therapeutic antibodies binding to distant parts of the flexible HER2, pertuzumab and trastuzumab (HTP). We highlight the strengths and limitations of current data processing software in dealing with various kinds of heterogeneities, particularly continuous conformational heterogeneity, and in describing the motions that can be extracted from our dataset. Our HTP structure provides a more detailed view than the one previously available for this ternary complex. This allowed us to pinpoint a previously overlooked loop in domain IV that may be involved both in binding of trastuzumab and in HER2 dimerization. This finding may contribute to explain the synergistic anticancer effect of the two antibodies. We further propose that the flexibility of the HTP complex, beyond the difficulties it causes for cryo-EM analysis, actually reflects regulation of HER2 signaling and its inhibition by therapeutic antibodies. Notably we obtain our best data with ultra-thin continuous carbon grids, showing that with current cameras their use to alleviate particle misdistribution is compatible with a protein complex of only 162 kDa. Perhaps most importantly, we provide here a dataset for such a smallish protein complex for further development of software accounting for continuous conformational heterogeneity in cryo-EM images.
摘要：
低温透射电子显微镜（cryo-EM）的单粒子分析对于结构预测仍然棘手的复合物特别有吸引力。如抗体-抗原复合物。在这里，我们获得了人表皮生长因子受体2（HER2）和来自两种不同治疗性抗体的抗原结合片段之间特别困难的复合物的详细结构，这些抗体结合到柔性HER2的远端部分，帕妥珠单抗和曲妥珠单抗（HTP）。我们强调了当前数据处理软件在处理各种异构性方面的优势和局限性，特别是连续的构象异质性，并描述可以从我们的数据集中提取的运动。我们的HTP结构提供了比以前可用于该三元复合物的视图更详细的视图。这使我们能够查明结构域IV中以前被忽视的环，该环可能参与曲妥珠单抗的结合和HER2二聚化。这一发现可能有助于解释两种抗体的协同抗癌作用。我们进一步建议HTP复合体的灵活性，除了它给低温EM分析带来的困难之外，实际上反映了HER2信号的调节及其治疗性抗体的抑制。值得注意的是，我们通过超薄连续碳网获得最佳数据，表明在当前相机中，它们用于减轻颗粒分布不均的用途与仅162kDa的蛋白质复合物兼容。也许最重要的是,我们在这里提供了一个如此小的蛋白质复合物的数据集，用于进一步开发软件，以解释低温EM图像中连续的构象异质性。
