Schematic representations of the precursors and assembled components of the branch junction. (A) Drawing of the inactive Arp2/3 complex and a standard actin filament. (B) Model of the branch junction. The following conformational changes are proposed to occur: (1) opening the nucleotide-binding clefts of mother filament subunits M2 and M4; (2) converting subunit M4 from a filament to a monomeric conformation; (3) converting Arp3 into filament conformation; (4) moving Arp2 tethered by ARPC5 next to Arp3 to form the first two subunits of the daughter filament; and (5) converting Arp2 into filament conformation.

Low resolution surface representation of the branch junction model. Actin subunits are shown in white or gray. M1–6 are subunits in the mother filament. D1 and D2 are the first two subunits in the daughter filament. Some of the actin and Arp subunits are indicated by numbers. (A) Three different views related by 90° clockwise rotations. (B) The three views of A showing the mother filament with areas of contact with subunits of the Arp2/3 complex color coded according to the subunit coloring in A. In the middle view, the Arp2/3 complex is viewed from the aspect of the mother filament (turned 180° clockwise from A) with points of mother filament contact marked in gray (subunits M2 and M4) and white (subunits M1, M3, M5, and M6). Bar, 5 nm.

Reconstruction of filament branch junction formed by the A. castellanii Arp2/3 complex. (A) Three views related by 90° clockwise rotations. The reconstruction was sharpened in Fourier space with 300 Ų and is displayed at a 100% mass contour level. All other density representations are from the original reconstruction. Numbers indicate three bridges of density between the two branches. (B) Stereo view of the fit of the branch junction model (α carbon trace) into the branch junction. Actin subunits are gray. Inset shows the crystal structure of the Arp2/3 complex with the mother filament position indicated by a gray rectangle. The color code for subunits of the Arp2/3 complex is used throughout: Arp2, red; Arp3, orange; ARPC1, green; ARPC2, cyan; ARPC3, magenta; ARPC4, blue; and ARPC5, yellow.

Electron tomography of actin filament branches mediated by the Arp2/3 complex. (A) Central slice showing several branches (boxes), individual Arp2/3 complexes (circles), and gold markers (arrowheads). (B and C) Enlarged view of individual complexes (B) and branches (C1–3). (D) Automatically segmented branch junctions show individual actin subunits and substructure in the junction. (E–G) Central slices through the averages of branch junctions from A. castellanii (E), cow (F), and budding yeast (G). (H) Composite of three x-y slices through a dual-axis cryotomogram. Because the branches are not contained within a single slice, we concatenated three slices for visualization purposes. (I) Central slices through the three branches marked in H after reorientation to coincide with the x-y plane. (J) Detail of a filament from H showing individual actin subunits after automatic segmentation. Bars: (A, C, D, H, and I) 20 nm; (G and J) 10 nm.

Fits of unmodified and modified crystal structures in the reconstruction of branch junctions. (A) Best fit of the Arp2/3 complex crystal structure. Arrows indicate mismatches with the reconstruction. The magenta and red arrows indicate the movement of ARPC3 and Arp2 upon remodeling. (B) Best fit of the remodeled Arp2/3 complex. (C and D) Comparisons between mother filament density in the branch reconstruction and a filament model viewed from the side opposite to the origin of the daughter filament. (C) Superimposition of the models fit to optimize the top portion of the reconstruction (orange) and the bottom portion of the reconstruction (dark pink). The gray arrow points to the area where a break in symmetry occurs at subunit M4. The pink and orange arrows indicate the movement necessary to fit the density. (D) Fit after replacing M4 with a monomer conformation. (E and F) Surface contours of the reconstruction at ∼70% of the mass. (E) Fit of the subdomains 2 of M2 (light pink) and M4 (dark gray) in the original filament conformation. The arrow points at subdomain 2 of actin subunit M4. (F) Improved fit with modified subdomains 2. (G) Close-up stereo view of the branch junction model inside the reconstruction displayed at the 100% mass contour level. The tethered helix of APC1 (green helix in the foreground) is near the groove between subdomains 1 and 3 of mother filament subunit M4.