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Uncapping of actin filaments is essential for driving polymerization and depolymerization dynamics from capping protein-associated filaments; however, the mechanisms of uncapping leading to rapid disassembly are unknown. Here, we elucidated the x-ray crystal structure of the actin/twinfilin/capping protein complex to address the mechanisms of twinfilin uncapping of actin filaments. The twinfilin/capping protein complex binds to two G-actin subunits in an orientation that resembles the actin filament barbed end. This suggests an unantic