Structure Determination of Human γ-Secretase.
Although the overall resolution limit for our previous cryo-EM structure of γ-secretase is 4.5 Å, the actual resolution in the TM region is considerably lower and reveals few features for the helices (31). To help identify the TMs in PS1, we fused T4 lysozyme to the amino terminus of PS1, hoping that this design may allow conclusive identification of PS1-TM1 even at a moderate resolution. To investigate the role of detergent on the integrity of γ-secretase, we replaced amphipols by digitonin. The resulting human γ-secretase exhibited excellent solution behavior (Fig. S1A) and robust protease activities toward the substrate APP C99 (Fig. S1 B and C). We imaged the γ-secretase with a K2 direct electron detector mounted on a Titan Krios electron microscope operating at 300 kV (Fig. 1).
Fig. 1.  Cryo-EM analysis of human γ-secretase. (A) A representative micrograph of the γ-secretase sample. An entire micrograph is shown. (B) Representative 2D classifications of the γ-secretase particle. The duck-shaped side views (identified by green dots) clearly reveal the position of the ECD (large head) and T4 lysozyme (small feet). The majority of the large duck body is formed by the nonspecifically bound detergents, whose contributions are gradually diminished at increasingly higher resolutions. (C) An overall view of the EM density for γ-secretase. The resolution is color-coded for different regions of γ-secretase. The relatively uniform resolution contrasts our previous study where the TM region has considerably lower resolutions (31). The EM density maps were generated in Chimera (46). (D) The overall resolution is estimated to be 4.32 Å on the basis of gold standard FSC criteria of 0.143. In total, 575,155 particles were selected from 3,312 micrographs for reference-free two-dimensional (2D) classification and subsequent three-dimensional (3D) classification (Fig. 1 A and B). After a second round of 3D classification, 177,207 particles from six classes were subjected to further autorefinement. The final EM density has an overall resolution of 4.32 Å on the basis of the gold standard Fourier Shell Correlation (FSC) criteria (Fig. 1 C and D). Nineteen TMs display unambiguous α-helical conformation, some with side-chain features (Fig. 2A and Figs. S2 and S3).
Fig. 2.  Overall structure of human γ-secretase. (A) An overall view of the EM density at 4.32-Å resolution. Densities for the four components of γ-secretase are color-coded: PS1 (blue), Pen-2 (yellow), Aph-1 (magenta), and nicastrin (green). Except TM2 of PS1, all other 19 TMs display clearly identifiable density. (B) Cartoon representation of the γ-secretase structure is shown in four perpendicular views. The four components are color-coded: PS1 (blue), Pen-2 (yellow), Aph-1 (magenta), and nicastrin (green). This coloring scheme is used in the other figures in this article. The 20 TMs assemble into a horseshoe-shaped structure. Notably, TM6 and TM7 of PS1, which harbor the two catalytic residues Asp257 and Asp385, are located on the convex side of the TM horseshoe. The structure figures were prepared using PYMOL (50). The central task—identification of PS1—was greatly facilitated by the presence of T4 lysozyme, which is attached to PS1-TM1 on the cytoplasmic side (Fig. 2A). The assignment of PS1-TM1, along with the predicted structural homology between PS1 and PSH, allows convenient identification of the other TMs (Fig. S2 A and B). Notably, PS1-TM2 has little density and belongs to the 20th TM beyond the 19 clearly observed TMs. Based on its connectivity to the ECD, the only TM in nicastrin was unambiguously assigned (Fig. S2 A and C). The seven TMs, located between nicastrin and PS1, were assigned to Aph-1 (Fig. S2 A and D). Finally, the remaining three TMs on the thin end of the TM horseshoe were attributed to Pen-2 (Fig. S2 A and E). The limited resolution only allowed interpretation of the TMs as a poly-Ala model. Importantly, however, the sequence homology between PS1 and PSH was used to generate a candidate atomic model for PS1.