Dataset Information

Heterogeneity of large macromolecular complexes revealed by 3D cryo-EM variance analysis.

ABSTRACT: Macromolecular structure determination by cryo-electron microscopy (EM) and single-particle analysis are based on the assumption that imaged molecules have identical structure. With the increased size of processed data sets, it becomes apparent that many complexes coexist in a mixture of conformational states or contain flexible regions. We describe an implementation of the bootstrap resampling technique that yields estimates of voxel-by-voxel variance of a structure reconstructed from the set of its projections. We introduce a highly efficient reconstruction algorithm that is based on direct Fourier inversion and that incorporates correction for the transfer function of the microscope, thus extending the resolution limits of variance estimation. We also describe a validation method to determine the number of resampled volumes required to achieve stable estimate of the variance. The proposed bootstrap method was applied to a data set of 70S ribosome complexed with tRNA and the elongation factor G. The proposed method of variance estimation opens new possibilities for single-particle analysis, by extending applicability of the technique to heterogeneous data sets of macromolecules and to complexes with significant conformational variability.

SUBMITTER: Zhang W

PROVIDER: S-EPMC2642923 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Heterogeneity of large macromolecular complexes revealed by 3D cryo-EM variance analysis.

Zhang Wei W Kimmel Marek M Spahn Christian M T CM Penczek Pawel A PA

Structure (London, England : 1993) 20081201 12

Macromolecular structure determination by cryo-electron microscopy (EM) and single-particle analysis are based on the assumption that imaged molecules have identical structure. With the increased size of processed data sets, it becomes apparent that many complexes coexist in a mixture of conformational states or contain flexible regions. We describe an implementation of the bootstrap resampling technique that yields estimates of voxel-by-voxel variance of a structure reconstructed from the set o ...[more]

PMID: 19081053

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Project description: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).

Dataset Information

Heterogeneity of large macromolecular complexes revealed by 3D cryo-EM variance analysis.

Publications

Heterogeneity of large macromolecular complexes revealed by 3D cryo-EM variance analysis.

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