Fig. 1.

Cryo-EM of the pure F-actin control. A typical segment used for image processing (480 Å long) is shown within the red box in the cryo-EM micrograph of pure F-actin (a). Actin filaments decorated with ABD2 of fimbrin (b) are noticeably thicker than pure actin filaments. (Scale bar: 500 Å.) Three-dimensional reconstruction of F-actin in the “canonical” state (c) was generated with the IHRSR method (17), using a subset (n = 10,986) of the total segments collected after sorting to exclude structural variability, such as tilted actin and segments where subdomain 2 was disordered. Actin subdomains are labeled with red numbers. To improve the fitting, the actin monomer was divided into three parts: SD1 (residues 2–33, 70–145, and 336–375), SD2 (34–69), and SD34 (146–335). These parts were fit as rigid bodies, and the corresponding symmetry was imposed to build a model filament (c, blue ribbons). To evaluate the similarity between the model and the actual reconstruction, the atomic model was filtered to 12 Å (d, transparent surface) so that it may be compared with the original volume (c, transparent surface). Our model is in agreement with the contacts between the protomers in the Holmes model (15). Residues that maintain longitudinal contacts (e) are 243–245 (blue) and 322–325 (cyan); 202–204 (yellow) and 286–289 (green); and 41–45 (red) and 166–169,375 (magenta). Lateral contacts are shown in f: 110–112 (magenta) bridges with (195–197 (cyan), while the tip of the hydrophobic loop (266–269, green) would contribute to the bridge of density with a hydrophobic pocket (166, 169, 171, 173, 285, and 289 shown in yellow, and 40–45 and 63–64 shown in red) formed by residues on the opposite strand. Because we have not rotated the loop from the body of the actin subunit, some of the red residues appear far from the loop.