期刊
ULTRAMICROSCOPY
卷 203, 期 -, 页码 170-180出版社
ELSEVIER
DOI: 10.1016/j.ultramic.2018.11.016
关键词
Single-particle cryo-EM; cisTEM; Frealign; Classification; Heterogeneity; Ribosome
类别
资金
- NIH [RO1 GM065056, P50 GM103368]
- Ohio State University
- [DP5 OD021396]
Single-particle electron cryo-microscopy and computational image classification can be used to analyze structural variability in macromolecules and their assemblies. In some cases, a particle may contain different regions that each display a range of distinct conformations. We have developed strategies, implemented within the Frealign and cisTEM image processing packages, to focus-classify on specific regions of a particle and detect potential covariance. The strategies are based on masking the region of interest using either a 2-D mask applied to reference projections and particle images, or a 3-D mask applied to the 3-D volume. We show that focused classification approaches can be used to study structural covariance, a concept that is likely to gain more importance as datasets grow in size, allowing the distinction of more structural states and smaller differences between states. Finally, we apply the approaches to an experimental dataset containing the HIV-1 Transactivation Response (TAR) element RNA fused into the large bacterial ribosomal subunit to deconvolve structural mobility within localized regions of interest, and to a dataset containing assembly intermediates of the large subunit to measure structural covariance.
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