ジャーナル: Front Mol Biosci / 年: 2021 タイトル: HEMNMA-3D: Cryo Electron Tomography Method Based on Normal Mode Analysis to Study Continuous Conformational Variability of Macromolecular Complexes. 著者: Mohamad Harastani / Mikhail Eltsov / Amélie Leforestier / Slavica Jonic / 要旨: Cryogenic electron tomography (cryo-ET) allows structural determination of biomolecules in their native environment (). Its potential of providing information on the dynamics of macromolecular ...Cryogenic electron tomography (cryo-ET) allows structural determination of biomolecules in their native environment (). 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 nucleosome conformational variability. HEMNMA-3D software is available freely (open-source) as part of ContinuousFlex plugin of Scipion V3.0 (http://scipion.i2pc.es).
EMPIAR-10679 (タイトル: Subtomograms of nucleosomes extracted from cryo-tomograms of Drosophila melanogaster embryos Data size: 666.3 MB Data #1: Unaligned subtomograms of nucleosomes extracted from in situ cryo-tomograms of Drosophila melanogaster embryonic brain [subtomograms])
凍結剤: OTHER / 詳細: high pressure freezer HPM010 (ABRA Fluid AG).
詳細
Drosophila melanogaster embryos were high-pressure frozen. Vitreous sections were cut with a nominal thickness of 75 nm and collected onto 200 mesh C-flat grids