Structural insights into actin filament recognition by commonly used cellular actin markers.

Cellular studies of filamentous actin (F‐actin) processes commonly utilize fluorescent versions of toxins, peptides, and proteins that bind actin. While the choice of these markers has been largely based on availability and ease, there is a severe dearth of structural data for an informed judgment in employing suitable F‐actin markers for a particular requirement. Here, we describe the electron cryomicroscopy structures of phalloidin, lifeAct, and utrophin bound to F‐actin, providing a comprehensive high‐resolution structural comparison of widely used actin markers and their influence towards F‐actin. Our results show that phalloidin binding does not induce specific conformational change and lifeAct specifically recognizes closed D‐loop conformation, i.e., ADP‐Pi or ADP states of F‐actin. The structural models aided designing of minimal utrophin and a shorter lifeAct, which can be utilized as F‐actin marker. Together, our study provides a structural perspective, where the binding sites of utrophin and lifeAct overlap with majority of actin‐binding proteins and thus offering an invaluable resource for researchers in choosing appropriate actin markers and generating new marker variants. Synopsis: Study of the cellular actin cytoskeleton relies on fluorescent probes based on toxins (phalloidin), peptides (LifeAct), or actin‐binding proteins (e.g. utrophin). Cryo‐EM structures and comparative analyses of these three probes bound to actin filaments offer guidance for their respective suitabilities. Phalloidin binding to actin filament does not induce actin conformational changes.LifeAct preferentially binds to the closed D‐loop conformation of actin filaments.The utrophin CH1 domain is sufficient for interaction with F‐actin. [ABSTRACT FROM AUTHOR]