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

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.