SimHS-AFMfit-MD: An Integrative Approach to Deciphering Alpha-Actinin Atomic Conformational Dynamics

Many molecular systems, such as intrinsically disordered proteins and flexible multi-domain complexes, are highly dynamic and often inaccessible to conventional X-ray crystallography or cryo-EM due to their conformational heterogeneity and flexibility. As a result, resolving their atomic-level dynamics remains a significant challenge. In this study, we present SimHS-AFMfit-MD, an integrative framework that combines high-speed atomic force microscopy (HS-AFM), molecular dynamics (MD) simulations, and AFMfit-based structural modeling to reconstruct dynamic protein conformations at atomic resolution. Using alpha-actinin, an actin crosslinking protein, as a challenging test system, we show that AFMfit guided by nonlinear normal mode analysis (AFMfit-NMA) enables accurate structural fitting, while guiding AFMfit with MD trajectories (AFMfit-MD) further enhances the flexible fitting performance, achieving closer agreement with unbiased all-atom MD simulation results. This strategy allows us to convert thousands of three-dimensional HS-AFM images into atomic-scale conformational ensembles, revealing the twisting and bending transitions underlying Ca{superscript 2}-bound and Ca{superscript 2}-unbound alpha-actinin. Together, our results establish a hybrid computational-experimental approach that bridges the spatial and, to some extent, temporal resolution gaps between simulation and imaging, paving the way for real-time visualization of protein conformational dynamics at the atomic scale. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/647477v3_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@147c1aforg.highwire.dtl.DTLVardef@1fd0036org.highwire.dtl.DTLVardef@118da3forg.highwire.dtl.DTLVardef@a062c3_HPS_FORMAT_FIGEXP M_FIG C_FIG