Subunit conformational variation within individual GroEL oligomers resolved by Cryo-EM

Abstract

Single-particle electron cryo-microscopy (cryo-EM) is an emerging tool for resolving structures of conformationally heterogeneous particles; however, each structure is derived from an average of many particles with presumed identical conformations. We used a 3.5-Å cryo-EM reconstruction with imposed D7 symmetry to further analyze structural heterogeneity among chemically identical subunits in each GroEL oligomer. Focused classification of the 14 subunits in each oligomer revealed three dominant classes of subunit conformations. Each class resembled a distinct GroEL crystal structure in the Protein Data Bank. The conformational differences stem from the orientations of the apical domain. We mapped each conformation class to its subunit locations within each GroEL oligomer in our dataset. The spatial distributions of each conformation class differed among oligomers, and most oligomers contained 10-12 subunits of the three dominant conformation classes. Adjacent subunits were found to more likely assume the same conformation class, suggesting correlation among subunits in the oligomer. This study demonstrates the utility of cryo-EM in revealing structure dynamics within a single protein oligomer.nderstanding the function of a molecular machine typically requires determination of the structural components at atomic or near-atomic resolution. Although X-ray crystallography can provide atomic details, the crystal lattice forces may unnaturally constrain the structure. Moreover, solution methods, such as NMR spectroscopy, can reveal short-range dynamic behavior in the absence of information on the entire complex (1, 2). Electron cryo-microscopy (cryo-EM) has made substantial progress toward achieving atomic resolution for a broad range of molecular machines by averaging thousands to millions of single-particle images that are assumed to have identical conformations (3-6).Cryo-EM has also been used to determine multiple structures of a single specimen preparation containing conformational and compositional heterogeneity (7,8). The focused classification and refinement approaches (7, 8) have been used for two primary purposes: sorting out localized structural heterogeneity among particles (9, 10) and improving feature resolvability of flexible domains through local refinement (11, 12). These molecular machines frequently are composed of multiple protein subunits that can generate correlated and/or uncorrelated motions (13), and no experimental technique has yet captured the atomic structures of individual subunits within a single molecular machine in solution. GroEL, Escherichia coli chaperonin (13,14), are composed of 14 chemically identical protomers (13,14) and have been structurally characterized by both X-ray crystallography (15, 16) and cryo-EM (5, 17-19). However, these studies have not revealed any heterogeneity among the protomers in each oligomer. Here we used a 3D classification strategy for cryo-EM data (10) to investigate structural heterogeneity among chemically identical subunits and their conformational v...

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