Automated Identification of Flexible Multivalent IDP-Bound Assemblies in Electron Micrographs
The quantitative characterization of compositional and conformational heterogeneity is an outstanding problem in structural biology and a focus of attention in the electron microscopy (EM) community. Here, we report automated analysis for a particular class of multivalent intrinsically disordered proteins (IDPs) bound to hub proteins, which exhibit not only a multiplicity of bound species but also continuous conformational flexibility. Such flexibility currently presents a significant challenge to standard methods of EM analysis, because “class averaging” effectively washes out the heterogeneity of primary interest. Alternatively, manual procedures can be used, but these are time-consuming and bias-prone. In the present work, we study a five-binding site IDP (Nup159) thought to bind in parallel duplex fashion to “hub” LC8 homodimers. We employ negative-stain EM (NSEM) because of its high contrast for single particle analysis and visualization of the small (∼20kDa) LC8 homodimer, although our approach should be applicable whenever there is sufficient contrast and a heterogeneous ensemble of oligomeric particles in EM micrographs. Procedurally, our scoring function identifies IDP-bound LC8 complexes based on chemical restraints imparted by the multivalent IDP scaffold, such as spacing and angles separating bound LC8 dimers. The results show a population distribution of oligomeric species (with 2, 3, 4 and 5 LC8 dimers), which is consistent with manually analyzed data. In addition, the automated procedure identifies several assemblies that were missed by initial manual analysis. Importantly, our automated approach allows for quantitative analysis of the ensemble of conformational states that are sampled by each of these classes, which had been obscured by traditional class averaging methods. The data also reveal the presence of oligomers with more than five LC8 dimers bound, suggesting out-of-register binding to the Nup159 duplex, which is a new finding.
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Mostofian, B., McFarland, R., Barbar, E. J., Reichow, S. L., & Zuckerman, D. M. (2020). Automated Identification of Flexible Multivalent IDP-Bound Assemblies in Electron Micrographs. Biophysical Journal, 118(3), 295a-296a.