Kolypetris, G.; Djurabekova, A.; Lasham, J.; Simsive, L.; Vonck, J.; Sharma, V.

Cryogenic-electron microscopy (cryo-EM) has revolutionized the field of protein structural biology. The structures of large membrane proteins are now routinely determined by cryo-EM to near atomic resolution. However, in the medium resolution range of cryo-EM maps (>~2 A), negatively charged sidechains of acidic residues are not well-resolved due to the negative electrostatic potential of the region. This may lead to incorrect sidechain models for residues like glutamic acid or aspartic acid that are central for proton transfer activity in various respiratory and photosynthetic enzymes. We previously proposed that the acidic residues with weak or non-existent cryo-EM density can be modeled to represent their low proton affinity conformations. Here, we tested this hypothesis on a larger data set of acidic amino acid residues in two high-resolution respiratory complex I structures. By using faster sidechain modeling and proton affinity prediction tools, we created a workflow that generates sidechain conformations of selected amino acid residues. We validated the sidechain conformation predictions by Q-score analysis and atomistic molecular dynamics simulations in different charged states. The proposed workflow provides a way to rapidly obtain sidechain conformations of acidic residues with weak cryo-EM densities and can be integrated into the existing cryo-EM modeling pipelines to speed up sidechain rotamer prediction.