Actin filaments function ubiquitously in essential cellular processes including motility, endocytosis, cell division, muscle contraction, and many others by scaffolding cells, generating protrusive forces, and serving as tracks for molecular motors. Consistent with its importance to all eukaryotic species, actin is the most abundant cytoplasmic protein and one of the most well-conserved evolutionarily. Actin is dynamic, in constant flux between its monomeric (G-actin) and filamentous (F-actin) forms. Some important mechanisms that regulate actin dynamics include: 1) biochemical properties of different actin isoforms and isoforms of actin-binding proteins like the F-actin-decorating protein tropomyosin (Tpm), 2) the asymmetry of the two F-actin ends, and 3) the interaction of F-actin ends with capping proteins. These features are essential for control of the assembly and disassembly of F-actin. To gain a better understanding of these mechanisms, we developed human cell expression and purification systems to produce native isoforms of actin and Tpm, performed biochemical experiments to test their unique properties, and solved cryogenic-electron microscopy structures of F-actin. Biochemical analyses along the multi-step expression procedures and mass spectrometry proteomics data demonstrate the fidelity of human cell-expressed actin and Tpm compared to tissue-purified counterparts. Expressed Tpm isoforms display different F-actin-binding affinities, indicative of their non-overlapping cellular roles. The structures of F-actin reveal the mode of binding for capping proteins and the mechanism for blocking monomer exchange. The work described here uncovers the biochemical and structural foundation for several mechanisms controlling F-actin dynamics.