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Abstract
Current challenges in the field of structural genomics point to the need for new tools and technologies for obtaining structures of macromolecular protein complexes. Here, we present an integrative computational method that uses molecular modelling, ion mobility-mass spectrometry (IM-MS) and incomplete atomic structures, usually from X-ray crystallography, to generate models of the subunit architecture of protein complexes. We begin by analyzing protein complexes using IM-MS, and by taking measurements of both intact complexes and sub-complexes that are generated in solution. We then examine available high resolution structural data and use a suite of computational methods to account for missing residues at the subunit and/or domain level. High-order complexes and sub-complexes are then constructed that conform to distance and connectivity constraints imposed by IM-MS data. We illustrate our method by applying it to multimeric protein complexes within the Escherichia coli replisome: the sliding clamp, (β2), the γ complex (γ3δδ′), the DnaB helicase (DnaB6) and the Single-Stranded Binding Protein (SSB4).
Highlights
Multi-protein complexes carry out various critical functions at almost every level of cellular organization, including ion transport, signaling, synthesis, waste management, and cell death [1]
Selection of Systems to Develop the Method To develop our computational method we focused on ion mobility-mass spectrometry (IM-MS)
Our goal is to reveal the topology of subunits in these oligomeric forms which assemble at high concentrations in solution [26]
Summary
Multi-protein complexes carry out various critical functions at almost every level of cellular organization, including ion transport, signaling, synthesis, waste management, and cell death [1]. Protein complexes comprise some of the most sought-after targets in molecular medicine [2] Due to their structural complexity and dynamic character, protein complexes can present a significant challenge to many of the ‘classical’ high-resolution structural biology tools (i.e. X-ray crystallography and Nuclear Magnetic Resonance (NMR) spectroscopy) [3,4]. Those tools often require large amounts of highly purified samples and many protein complexes are too polydisperse or too scarce within the cellular matrix for structural characterisation. The number of complete high-resolution structures of multi-subunit complexes deposited in structural databases remains relatively low [5]
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