Refinement of High-resolution Cryo-EM Structure of hERG: What can we Expect?

Abstract

Cryo-EM method has emerged as a major structure solving tool in structural biology in recent years. One of the biggest advantages of the Cryo-EM method is its ability to solve large biomolecular complexes in their native environment. However, even high-resolution Cryo-EM structures contain poor side chain conformations and interatomic clashes. This might lead to the loss of many physiologically relevant interactions. For such structures, although obtained at high-resolution, further structural refinement is necessary for establishing structure-function relationships. We present such a case for the human ether-a-go-go-related gene (hERG) encoded voltage gated potassium channel (KCNH2 or Kv11.1). The Kv11.1 open state structure was solved at 3.8 Å. However, many relevant salt bridges were missing in the Voltage Sensing Domain (VSD). Using Molecular Dynamics Flexible Fitting (MDFF) technique, we further refined the open state of the Kv11.1 structure which underwent microsecond long simulation before fitting. Extracted structures from the MD simulation were fitted to the Cryo-EM map. During the fitting process, we tested different environments, including vacuum, implicit solvent and an explicit bilayer. Our comparison of the final fitted structures indicate that the bilayer fitted MDFF structure is the most reasonable one when compared to the existing biochemical and electrophysiology data as this structure contains most of the missing salt bridges in the VSD. This result suggests that molecular dynamics simulations, based on a detailed force field and extensive sampling, can provide additional structural information that enables more detailed mechanistic investigations.