Determining the 3D Topologies of Heteromeric Protein Assemblies by a Mass-Spectrometry Based Hybrid Approach

Abstract

Describing, understanding, and modulating the function of the cell require elucidation of the networks and structure of its macromolecular assemblies. Here, we describe an integrative approach to determine the topologies of heteromeric assemblies using structural information derived from native mass spectrometry (MS), proteomics and chemical cross-linking MS. The method was developed and further assessed for robustness and accuracy using a benchmark of five hypothetical assemblies with simulated data and two assemblies with previously published MS data. With this benchmark in hand, we purify and characterize the yeast eukaryotic initiation factor 3 (eIF3) complex whose complete 3D topology has not been previously defined. Using a combination of MS-based data, we establish sub-stoichiometric binding of eIF5 and derive a high-likelihood structural model for the five subunit eIF3 complex. Our results further reveal two interaction modules within the eIF3 and are in accord with its role as a scaffold for other initiation factors. In conclusion, here we highlight the utility of a MS-based integrative approach for modelling complexes with unknown interactions and topology. The corresponding computational algorithm is implemented in the open source Integrative Modeling Platform (IMP).View Large Image | View Hi-Res Image | Download PowerPoint Slide