Actin has maintained an exquisite degree of sequence conservation over large evolutionary distances for reasons that are not understood. The desire to explain phenomena from muscle contraction to cytokinesis in mechanistic detail has driven the generation of an atomic model of the actin filament (F-actin). Here we use electron cryomicroscopy to show that frozen-hydrated actin filaments contain a multiplicity of different structural states. We show (at ∼10 Å resolution) that subdomain 2 can be disordered and can make multiple contacts with the C terminus of a subunit above it. We link a number of disease-causing mutations in the human ACTA1 gene to the most structurally dynamic elements of actin. Because F-actin is structurally polymorphic, it cannot be described using only one atomic model and must be understood as an ensemble of different states.