Abstract
Structure determination using cryo-electron microscopy (cryo-EM) medium-resolution density maps is often facilitated by flexible fitting. Avoiding overfitting, adjusting force constants driving the structure to the density map, and emulating complex conformational transitions are major concerns in the fitting. To address them, we develop a new method based on a three-step multi-scale protocol. First, flexible fitting molecular dynamics (MD) simulations with coarse-grained structure-based force field and replica-exchange scheme between different force constants replicas are performed. Second, fitted Cα atom positions guide the all-atom structure in targeted MD. Finally, the all-atom flexible fitting refinement in implicit solvent adjusts the positions of the side chains in the density map. Final models obtained via the multi-scale protocol are significantly better resolved and more reliable in comparison with long all-atom flexible fitting simulations. The protocol is useful for multi-domain systems with intricate structural transitions as it preserves the secondary structure of single domains.
Highlights
The technical advances in single-particle cryo-electron microscopy (cryo-EM) result in a rapid escalation of the number of available density maps at increasingly higher resolutions recently crossing the 1.3 Å barrier (Yip et al, 2020) (Nakane et al, 2020)
We focus on medium resolution density maps, i.e., around 5-8 Å, since the flexible fitting is generally useful for such maps
One amino acid is represented by a single bead at their Cα atom position, and the beads interact via the structure-based potential that is constructed from the native structure
Summary
The technical advances in single-particle cryo-EM result in a rapid escalation of the number of available density maps at increasingly higher resolutions recently crossing the 1.3 Å barrier (Yip et al, 2020) (Nakane et al, 2020). At near-atomic resolution (typically 3-5 Å), the structural details visible in the density give enough confidence for building the models de novo, even though the completeness and accuracy of such models would be lower than those based on atomic resolution density maps (Wang et al, 2015) (Zivanov et al, 2018). The density maps do not always reach so high resolutions due to intrinsic flexibility or fluctuation of biomolecules, and the methods other than de novo building are often required in such cases
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