SignificanceActin polymerization is a divalent cation-dependent process. Here we identify a cation binding site on the surface of actin in a 2.0-Å resolution X-ray structure of actin and find evidence of three additional sites in published high-resolution structures. These cations are stable in molecular dynamics (MD) simulations of the filament, suggesting a functional role in polymerization or filament rigidity. Polymerization activates the ATPase activity of the incorporating actin protomers. Careful analysis of water molecules that approach the ATP in the MD simulations revealed Gln137-activated water to be in a suitable position in F-actin, to initiate attack for ATP hydrolysis, and its occupancy was dependent on bound cations.

Structural biology has experienced several transformative technological advances in recent years. These include: development of extremely bright Xray sources (microfocus synchrotron beamlines and free electron lasers) and the use of electrons to extend protein crystallography to ever decreasing crystal sizes; and an increase in the resolution attainable by cryo-electron microscopy. Here we discuss the use of these techniques in general terms and highlight their application for biological filament systems, an area that is severely underrepresented in atomic resolution structures. We assemble a model of a capped tropomyosin-actin minifilament to demonstrate the utility of combining structures determined by different techniques. Finally, we survey the methods that attempt to transform high resolution structural biology into more physiological environments, such as the cell. Together these techniques promise a compelling decade for structural biology and, more importantly, they will provide exciting discoveries in understanding the designs and purposes of biological machines.

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