Carlos F. Rodríguez, Mohinder Pal, Hugo Muñoz-Hernandez, Laurence H. Pearl, and Oscar Llorca
The number of high-resolution structures of protein complexes obtained using cryo-electron microscopy (cryo-EM) is increasing rapidly. Cryo-EM maps of large macromolecular complexes frequently contain regions resolved at different resolution levels, and modeling atomic structures de novo can be difficult for domains determined at worse than 5 Å in the absence of atomic information from other structures. Here we describe the details and step-by-step decisions in the strategy we followed to model the RUVBL2-binding domain (RBD), a 14 kDa domain at the C-terminus of RNA Polymerase II associated protein 3 (RPAP3) for which atomic information was not available. Modeling was performed on a cryo-EM map at 4.0–5.5 Å resolution, integrating information from secondary structure predictions, homology modeling, restraints from cross-linked mass spectrometry, and molecular dynamics (MD) in AMBER. Here, we compare our model with the structure of RBD determined by NMR to evaluate our strategy. We also perform new MD simulations to describe important residues mediating the interaction of RBD with RUVBL2 and analyze their conservation in RBD homologous domains. Our approach and its evaluation can serve as an example to address the analysis of medium resolution regions in cryo-EM maps.
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