PROJECT 4: INTEGRATIVE MODELING OF ABC TRANSPORTERS SUMMARY We aim to determine the structures, thermodynamics, and functional dynamics of the key snapshot states in the conformational cycle of prokaryotic and eukaryotic ABC transporters, with a broader goal to contribute towards understanding the function of ABC transporters in general. Only sparse information about these structures is currently available, due to sample unavailability and limitations of any single structure determination technique when applied to the compositionally and conformationally heterogeneous samples. To overcome these challenges, integrative structure determination that simultaneously considers data from a variety of different experimental methods will be adapted and used. We will rely on our Integrative Modeling Platform (IMP), an open source software package that provides programmatic support for developing and distributing integrative structure modeling protocols. Here, we will use IMP to facilitate an iterative experimental and computational process for constructing near-atomic resolution models of the functional states of the transporters. The building of structural models is generally best cast as a computational optimization problem where information about the modeled system is encoded into spatial restraints comprising a scoring function used to evaluate candidate models. Our formulation of the scoring function using a Bayesian approach will allow us to model multiple states simultaneously, based on sparse, noisy, and ambiguous data generated from heterogeneous samples. The spatial restraints on the structures will be derived from all relevant data generated by the proposed Program and others, including datasets from X-ray crystallography, near-atomic resolution cryo-electron microscopy (EM), fluorescence resonance energy transfer (FRET) spectroscopy, double electron-electron resonance (DEER) spectroscopy, small angle X-ray scattering (SAXS), as well as chemical and cysteine cross-linking experiments. With snapshot structures in hand, we will also attempt to define the free energy and entropy differences between the functional states, by counting the frequency of each state in EM images. Finally, we will apply both simplified simulations and atomistic molecular dynamics in pilid environment to interpolate between the snapshots, and thus characterize the entire transport cycle at atomic resolution. Our integrative approach, honed here on key example transporters, will be applicable to many other large complexes of membrane proteins.
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