SimHS-AFMfit-MD: An Integrative Approach for Inferring Alpha-Actinin Atomic Conformational Dynamics.

Abstract

Many molecular systems, including intrinsically disordered proteins and flexible multidomain complexes, exhibit significant conformational heterogeneity and flexibility, making them difficult to study with conventional methods like X-ray crystallography or cryo-EM. To address this challenge, we introduce SimHS-AFMfit-MD, an integrative framework combining high-speed atomic force microscopy (HS-AFM), molecular dynamics (MD) simulations, and AFMfit-based structural modeling to infer dynamic protein conformations at atomic resolution. Using alpha-actinin, an actin cross-linking protein, as a model system, we demonstrate that nonlinear normal-mode analysis (AFMfit-NMA) enhances the accuracy of structural fitting. Additionally, guiding AFMfit with MD trajectories (AFMfit-MD) significantly improves fitting performance, aligning closely with unbiased all-atom MD simulations. This method converts thousands of 3D HS-AFM images into atomic-scale conformational ensembles, revealing key transitions between Ca²⁺-bound and Ca²⁺-unbound states of alpha-actinin. Our results showcase a hybrid computational-experimental approach that bridges simulation and imaging approaches, enabling real-time visualization of protein dynamics at the atomic scale.