FIGURE 1:

3D averaging of SMLM images of the nuclear pore complex. (A) Schematic representation of the NPC indicating the positions of the proteins imaged in this study. (B) Schematic representation of the 3D SMLM averaging pipeline. From a dual-color 3D SMLM image stack of the lower surface of the nucleus of a U2OS cell, endogenously expressing a SNAP-Nup labeled with BG-AF647 and ELYS visualized with antibodies coupled to CF680, individual pores were automatically picked and an iterative 3D alignment process was applied to the ELYS channel, imposing an eightfold rotational symmetry. The same transformation was then applied to register the NOI in the other channel to preserve the spatial relationship between the two labeled proteins. The final reference image was visualized as an intensity-rendered 3D reconstruction.