Data_Sheet_1_HEMNMA-3D: Cryo Electron Tomography Method Based on Normal Mode Analysis to Study Continuous Conformational Variability of Macromolecular Complexes.pdf

Cryogenic electron tomography (cryo-ET) allows structural determination of biomolecules in their native environment (in situ). Its potential of providing information on the dynamics of macromolecular complexes in cells is still largely unexploited, due to the challenges of the data analysis. The crowded cell environment and continuous conformational changes of complexes make difficult disentangling the data heterogeneity. We present HEMNMA-3D, which is, to the best of our knowledge, the first method for analyzing cryo electron subtomograms in terms of continuous conformational changes of complexes. HEMNMA-3D uses a combination of elastic and rigid-body 3D-to-3D iterative alignments of a flexible 3D reference (atomic structure or electron microscopy density map) to match the conformation, orientation, and position of the complex in each subtomogram. The elastic matching combines molecular mechanics simulation (Normal Mode Analysis of the 3D reference) and experimental, subtomogram data analysis. The rigid-body alignment includes compensation for the missing wedge, due to the limited tilt angle of cryo-ET. The conformational parameters (amplitudes of normal modes) of the complexes in subtomograms obtained through the alignment are processed to visualize the distribution of conformations in a space of lower dimension (typically, 2D or 3D) referred to as space of conformations. This allows a visually interpretable insight into the dynamics of the complexes, by calculating 3D averages of subtomograms with similar conformations from selected (densest) regions and by recording movies of the 3D reference's displacement along selected trajectories through the densest regions. We describe HEMNMA-3D and show its validation using synthetic datasets. We apply HEMNMA-3D to an experimental dataset describing in situ nucleosome conformational variability. HEMNMA-3D software is available freely (open-source) as part of ContinuousFlex plugin of Scipion V3.0 (http://scipion.i2pc.es).

冷冻电子断层扫描（Cryogenic electron tomography，cryo-ET）可在天然环境（原位）下解析生物大分子的结构。受限于数据分析的诸多挑战，其用于获取细胞内大分子复合物动态信息的潜力仍未得到充分挖掘。拥挤的细胞环境与复合物持续发生的构象变化，大幅增加了解析数据异质性的难度。本文提出HEMNMA-3D方法，据我们所知，这是首个针对复合物连续构象变化开展冷冻电子亚断层图（subtomogram）分析的方法。HEMNMA-3D将柔性三维参考模型（原子结构或电子显微镜密度图）的弹性与刚体三维-三维迭代比对相结合，以匹配每个亚断层图中复合物的构象、取向与位置。其中弹性比对结合了分子力学模拟（三维参考模型的简正模式分析（Normal Mode Analysis））与实验亚断层图数据分析；刚体重比对则针对冷冻电子断层扫描有限倾角导致的缺失楔效应（missing wedge）进行校正。通过比对得到的各亚断层图中复合物的构象参数（简正模式振幅）将被处理，以在低维空间（通常为二维或三维，即构象空间（space of conformations））中可视化构象分布。通过从选定的（最密集）区域选取具有相似构象的亚断层图计算三维平均，并记录三维参考模型沿穿过最密集区域的选定轨迹的位移动画，可实现对复合物动态的可视化可解释解析。本文详细阐述了HEMNMA-3D方法，并利用合成数据集验证了其有效性。我们将HEMNMA-3D应用于描述原位核小体构象变异的实验数据集。HEMNMA-3D软件作为Scipion V3.0的ContinuousFlex插件的一部分免费开源，相关获取地址为http://scipion.i2pc.es。