SDS/PAGE analyses of FAS purified from the breast cancer cell line ZR75–1. (A) Various fractions were subjected to SDS/PAGE analysis (4–12% gradient), and the gel then was stained with Coomassie blue. Lane 1, soluble extract (25 μg); lanes 2, 3, and 4, HiLoad 26/10 Q-Sepharose fractions 29, 30–35, and 36 (10 μg of each fraction); lane 5, Superose-6 fraction (2 μg); lane 6, protein standard (molecular masses: 217, 123, 71, and 48 kDa); lanes 7, 8, 9, and 10, UNO-Q-6 fractions 56–59 (0.6 μg each). (B) For electron cryomicroscopic examination, the purified FAS (lane 5, 0.6 μg) was applied to an UNO-Q-6 column and fraction 3 (0.6 μg) was analyzed (lane 3). Lane 4 is a protein standard.

X-ray solution scattering intensity curve of FAS collected at the Stanford Synchrotron Radiation Laboratory on beamline BL 4-2. The scattering curve exhibits a shoulder at 1/70 Å⁻¹, which is characteristic for a dimeric particle. The curve was obtained by combining two separate scattering curves as explained in Materials and Methods.

Typical micrograph of FAS embedded in vitreou ice and recorded at 2.7-μm defocus. The image shown was median-filtered by using a 3 × 3-pixel window to facilitate identification and selection of the molecules. For clarity, several particle images that were selected for image processing have been boxed out. These particles show FAS in different views (e.g. double-arched and dumbbell-shaped). The accompanying close-to-focus micrograph of this area was used to select particles from for image processing. (Bar, 500 Å.)

3D reconstruction of FAS obtained with (A–D) and without (E–H) applying C₂ symmetry. The surface renderings were done at 100% of the molecular mass assuming 1.23 ų/Da. From left to right, different views of the molecule are shown, i.e., top view (A and E), side view (B and F), end-on view (C and G), and the view corresponding to the double-arch view visible in Fig. 3 (D and H). Individual domains of the subunits have been indicated as I–III (A and E). The far-right bottom view shows the two clefts formed by the monomers labeled as narrow (N) and wide (W). Proposed locations of the active sites in FAS are indicated by the two gray semitransparent oval regions.

Excerpts from the refinement of FAS using EMAN. Shown are (from left to right) projections of the 3D model along with the corresponding class averages as well as three particle images from each class. The particle images displayed differ by an in-plane rotation. Note that during the alignment, however, this difference is taken care of. The rows show top, side, and end-on views of FAS; the Bottom shows the characteristic view of two parallel arcs as seen in Fig. 2.